Nucleic acid amplification

ABSTRACT

Disclosed is a method of producing replicates of sample nucleic acids. The method can include providing an insoluble support comprising attached oligonucleotides, annealing sample nucleic acids to the attached oligonucleotides; constructing template nucleic acids by extending the attached oligonucleotides using a polymerase; and transcribing the template nucleic acids to produce RNA replicates of the sample nucleic acids The attached oligonucleotides comprise a promoter sequence and a target annealing sequence, and (2) the proximal end of the promoter sequence is spaced from the insoluble support by a predetermined distance.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of, claimspriority to, and incorporates by reference U.S. application Ser. No.10/341,199, filed on Jan. 10, 2003.

BACKGROUND

[0002] Genetic information can be analyzed for a number of applications,including medical diagnosis, genotyping, and forensics. The highthroughput analysis of nucleic acid samples is facilitated by nucleicacid amplification.

[0003] A variety of techniques can be used for nucleic acidamplification. The polymerase chain reaction (PCR; Saiki, et al. (1985)Science 230, 1350-1354) and ligase chain reaction (LCR; Wu. et al.(1989) Genomics 4, 560-569; Barringer et al. (1990), Gene 1989, 117-122;F. Barany. 1991, Proc. Natl. Acad. Sci. USA 1988, 189-193) utilizecycles of varying temperature to drive rounds of synthesis.Transcription-based methods utilize RNA synthesis by RNA polymerases toamplify nucleic acid (U.S. Pat. Nos. 6,066,457, 6,132,997, and5,716,785; Sarkar et al., Science (1989) 244:331-34; Stofler et al.,Science (1988) 239:491). NASBA (U.S. Pat. Nos. 5,130,238; 5,409,818; and5,554,517) utilizes cycles of transcription, reverse-transcription, andRNaseH-based degradation to amplify a DNA sample. Still otheramplification methods include rolling circle amplification (RCA; U.S.Pat. Nos. 5,854,033 and 6,143,495) and strand displacement amplification(SDA; U.S. Pat. Nos. 5,455,166 and 5,624,825).

SUMMARY

[0004] The invention is based, in part, on the discovery of atranscription-based method for amplifying nucleic acids. The method usesan immobilized oligonucleotide that includes a promoter and a targetannealing region. The promoter is spaced by a predetermined distancefrom the insoluble support, e.g., by a distance that enables efficienttranscription of the target sequence. The predetermined distance candepend on the particular reaction conditions, for example, the type ofinsoluble support, promoter, and attachment between the oligonucleotideand the support.

[0005] Accordingly, in one aspect, the invention features a method thatincludes: providing an insoluble support including attachedoligonucleotides, wherein (1) the attached oligonucleotides include apromoter sequence and a target annealing sequence, and (2) the proximalend of the promoter sequence is spaced from the insoluble support by adistance greater than 5 nm or 10 nm, annealing sample nucleic acids tothe attached oligonucleotides; constructing template nucleic acids byextending the attached oligonucleotides using a polymerase; andtranscribing the template nucleic acids to produce RNA replicates of thesample nucleic acids. For example, the distance can be between 10 to 150nm or 10 and 50 nm.

[0006] In one embodiment, the attached oligonucleotides each include aligand 5′ of the promoter (e.g., at the 5′ terminus), are attached tothe insoluble support by a non-covalent interaction (e.g., aligand/ligand-binding protein interaction), and the proximal end of thepromoter is between 5-30, 6-18, or 6-12 nucleotides from the ligand,e.g., from the 5′ terminus. For example, the attached oligonucleotidesinclude a biotin moiety (e.g., at the 5′ terminus), are attached to theinsoluble support by a biotin/biotin-binding protein interaction (e.g.,avidin or streptavidin), and the proximal end of the promoter is atleast 5 nucleotides, e.g., between 5-30 nucleotides from the biotinmoiety.

[0007] In another embodiment, the attached oligonucleotides are attachedto the insoluble support by a polyethylene glycol linker that has atleast 8 units or between 8-20 or 8-16 units, by a chemical linker havinga main chain length including the same number of main chain atoms as thepolyethylene glycol linker or having the same physical length as thepolyethylene glycol linker.

[0008] In another embodiment, the attached oligonucleotides arecovalently attached at their 5′ terminus, and the proximal end of thepromoter is between 12-50, 20-35, or 23-28 nucleotides from the 5′terminus of each of the attached oligonucleotides.

[0009] The sample nucleic acids include RNA, DNA, PNA, or other nucleicacid molecules.

[0010] The constructing can include extending the attachedoligonucleotide using an RNA-directed DNA polymerase to produce anextended stranded and synthesizing a DNA strand complementary to theextended strand to produce complementary strands, e.g., by a methoddescribed herein. The attached and complementary strands anneal, therebyproviding the template nucleic acids.

[0011] The method can include joining an adaptor that includes a tagsequence to the double-stranded template. The adaptor can include apromoter sequence, e.g., a prokaryotic promoter sequence. For example,the adaptor includes double-stranded DNA.

[0012] The promoter sequence can be a prokaryotic promoter sequence,e.g., a bacteriophage promoter sequence, e.g., a T7, T3, or SP6 promotersequence. In one embodiment, at least some of the attachedoligonucleotides include a promoter and a homopolymeric T tract. Theseattached oligonucleotides can further include a 3′ terminal A, G, or C.

[0013] The distance between the proximal end of the promoter sequenceand the insoluble support can be sufficient to enable at least 2, 4, 8,or 16 times the yield (e.g., between 2 and 32 times) of replicate RNAsas obtained using a distance of less than 2 nm between the proximal endof the promoter sequence and the insoluble support.

[0014] In another aspect, the invention features a method that includes:providing an insoluble support including attached template nucleic acids(e.g., covalently or non-covalently attached), wherein (1) each attachedtemplate nucleic acids include a promoter sequence and a targetsequence, and (2) the proximal end of the promoter sequence is spacedfrom the insoluble support by a predetermined distance; and transcribingthe template nucleic acids to produce RNA replicates of the samplenucleic acids. For example, the distance between the proximal end of thepromoter sequence and insoluble support can be between 10 to 150 nm or10 and 50 nm.

[0015] In one embodiment, the attached template nucleic acids eachinclude a ligand 5′ of the promoter (e.g., at the 5′ terminus), areattached to the insoluble support by a non-covalent interaction (e.g., aligand/ligand-binding protein interaction), and the proximal end of thepromoter is between 5-30, 6-18, or 6-12 nucleotides from the ligand,e.g., from the 5′ terminus. For example, the attached templates includea biotin moiety (e.g., at the 5′ terminus), are attached to theinsoluble support by a biotin/biotin-binding protein interaction (e.g.,avidin or streptavidin), and the proximal end of the promoter is atleast 5 nucleotides, e.g., between 5-30 nucleotides from the biotinmoiety.

[0016] In another embodiment, the attached template nucleic acids areattached to the insoluble support by a polyethylene glycol linker thathas at least 8 units or between 8-20 or 8-16 units, by a chemical linkerhaving a main chain length including the same number of main chain atomsas the polyethylene glycol linker or having the same physical length asthe polyethylene glycol linker.

[0017] In another embodiment, the attached template nucleic acids arecovalently attached at their 5′ terminus, and the proximal end of thepromoter is between 12-50, 20-35, or 23-28 nucleotides from the 5′terminus of each of the attached template nucleic acids.

[0018] The template nucleic acids can further include a second promoterpositioned to transcribe a nucleic acid segment located between thefirst and second promoters. Each is configured to transcribe a strand ofthe nucleic acid segment such that both strands of the nucleic acidsegment are transcribed. This method includes transcribing the templatenucleic acid using the first and second promoters to produce RNAcomplementary to each strand, and recovering double-stranded RNA for thenucleic acid segment.

[0019] The template nucleic acids can correspond to nucleic acids in abiological sample, e.g., a sample of nucleic acids obtained from a cell,e.g., from a culture cell, tissue, free-living cell or organism. Thetemplate nucleic acids can include regions that represent nucleic acidsin the sample in comparable proportions. For example, the templatenucleic acids can correspond to eukaryotic mRNAs, genomic DNAs, and soforth.

[0020] In one embodiment, a plurality of the template nucleic acids eachcomprises a common adaptor sequence at their respective distal ends. Theadaptor sequence can include a promoter sequence.

[0021] In another aspect, the invention features a method of archiving asample of complex nucleic acids. The method includes: providing a firstinsoluble support having 5′ attached oligonucleotide, wherein theattached oligonucleotide includes a promoter sequence that is at least 4nm from the insoluble support; annealing a complex sample that includessample nucleic acids to the insoluble support; and producing templatenucleic acids immobilized on the insoluble support that each include atleast a segment of the sample nucleic acids, the immobilized templatesrepresenting the composition of the sample nucleic acids; transcribingthe template nucleic acids from the insoluble support; archiving theinsoluble support; and transcribing the template nucleic acids from theinsoluble support. For example, the distance can be between 10 to 150 nmor 10 and 50 nm.

[0022] In one embodiment, the attached oligonucleotides each include aligand 5′ of the promoter (e.g., at the 5′ terminus), are attached tothe insoluble support by a non-covalent interaction (e.g., aligand/ligand-binding protein interaction), and the proximal end of thepromoter is between 5-30, 6-18, or 6-12 nucleotides from the ligand,e.g., from the 5′ terminus. For example, the attached oligonucleotidesinclude a biotin moiety (e.g., at the 5′ terminus), are attached to theinsoluble support by a biotin/biotin-binding protein interaction (e.g.,avidin or streptavidin), and the proximal end of the promoter is atleast 5 nucleotides, e.g., between 5-30 nucleotides from the biotinmoiety.

[0023] In another embodiment, the attached oligonucleotides are attachedto the insoluble support by a polyethylene glycol linker that has atleast 8 units or between 8-20 or 8-16 units, by a chemical linker havinga main chain length including the same number of main chain atoms as thepolyethylene glycol linker or having the same physical length as thepolyethylene glycol linker.

[0024] In another embodiment, the attached oligonucleotides arecovalently attached at their 5′ terminus, and the proximal end of thepromoter is between 12-50, 20-35, or 23-28 nucleotides from the 5′terminus of each of the attached oligonucleotides.

[0025] The template nucleic acids can correspond to nucleic acids in abiological sample, e.g., a sample of nucleic acids obtained from a cell,e.g., from a culture cell, tissue, free-living cell or organism. Thetemplate nucleic acids can include regions that represent nucleic acidsin the sample in comparable proportions. For example, the templatenucleic acids can correspond to eukaryotic mRNAs, genomic DNAs, and soforth.

[0026] The invention also features insoluble supports and immobilizedoligonucleotides described herein, e.g., including one or more featuresdescribed herein. The insoluble supports can include template nucleicacids, e.g., including features acquired from a sample described herein.

[0027] In another aspect, the invention features an insoluble supportthat includes a plurality of attached oligonucleotides, wherein (1) theattached oligonucleotides include a prokaryotic promoter sequence and atarget annealing sequence, (2) the target annealing sequence is 3′ ofthe promoter, (3) the oligonucleotide has an extendable 3′ terminus; and(4) the proximal end of the promoter sequence is spaced from theinsoluble support by a distance greater than 10 nm, In one embodiment,the oligonucleotides are less than 80 nucleotides in length.

[0028] In one embodiment, each target annealing sequence of theplurality is the same and the target annealing sequence can anneal to aplurality of different target sequences. For example, the targetannealing sequence can include a poly-thymidine tract. Each targetannealing sequence of the plurality can include a poly-thymidine tractand a terminal 3′ A, G, or C.

[0029] In one embodiment, target nucleic acids are annealed to thesupport, e.g., target nucleic acids from a sample described herein.

[0030] In one embodiment, the attached oligonucleotides each include aligand 5′ of the promoter (e.g., at the 5′ terminus), are attached tothe insoluble support by a non-covalent interaction (e.g., aligand/ligand-binding protein interaction), and the proximal end of thepromoter is between 5-30, 6-18, or 6-12 nucleotides from the ligand,e.g., from the 5′ terminus. For example, the attached oligonucleotidesinclude a biotin moiety (e.g., at the 5′ terminus), are attached to theinsoluble support by a biotin/biotin-binding protein interaction (e.g.,avidin or streptavidin), and the proximal end of the promoter is atleast 5 nucleotides, e.g., between 5-30 nucleotides from the biotinmoiety.

[0031] In another embodiment, the attached oligonucleotides are attachedto the insoluble support by a polyethylene glycol linker that has atleast 8 units or between 8-20 or 8-16 units, by a chemical linker havinga main chain length including the same number of main chain atoms as thepolyethylene glycol linker or having the same physical length as thepolyethylene glycol linker.

[0032] In another embodiment, the attached oligonucleotides arecovalently attached at their 5′ terminus, and the proximal end of thepromoter is between 12-50, 20-35, or 23-28 nucleotides from the 5′terminus of each of the attached oligonucleotides.

[0033] In another aspect, the invention features an insoluble supportthat includes attached template nucleic acids, wherein (1) each attachedtemplate nucleic acids include a prokaryotic promoter sequence, a targetsequence, and a ligand (2) for each template nucleic acid, the promoteris located between the target sequence and ligand, (3) the templatenucleic acids can be transcribed to produce RNA copies of eachrespective target sequence, (4) the ligand is bound to a ligand-bindingprotein immobilized on the support, and (5) the proximal end of thepromoter sequence is spaced from the ligand between 5 and 30nucleotides.

[0034] In a related aspect, the support includes attached templatenucleic acids, wherein (1) each attached template nucleic acids includea prokaryotic promoter sequence and a target sequence, (2) for eachtemplate nucleic acid, the promoter is located between the targetsequence and the site that attaches the template nucleic acid to thesupport, (3) the template nucleic acids can be transcribed to produceRNA copies of each respective target sequence, and (4) the templatenucleic acids is spaced from the support by a nucleotide-free linkerthat includes an identical number of main chain atoms as a polyethyleneglycol linker that has at least 8 units or between 8 and 16 units.

[0035] In another related aspect, the support includes attached templatenucleic acids, wherein (1) each attached template nucleic acids includea prokaryotic promoter sequence and a target sequence, (2) for eachtemplate nucleic acid, the promoter is located between the targetsequence and the site that attaches the template nucleic acid to thesupport, (3) the template nucleic acids can be transcribed to produceRNA copies of each respective target sequence, and (4) the attachedtemplate nucleic acids are covalently attached to the support, and theproximal end of the promoter is between 12 and 50 nucleotides from the5′ terminus of each of the oligonucleotides.

[0036] The template nucleic acids can correspond to nucleic acids in abiological sample, e.g., a sample of nucleic acids obtained from a cell,e.g., from a culture cell, tissue, free-living cell or organism. Thetemplate nucleic acids can include regions that represent nucleic acidsin the sample in comparable proportions. For example, the templatenucleic acids can correspond to eukaryotic mRNAs, genomic DNAs, and soforth.

[0037] In one embodiment, a plurality of the template nucleic acids eachcomprises a common adaptor sequence at their respective distal ends. Theadaptor sequence can include a promoter sequence.

[0038] In another aspect, the invention features a method that includes:cleaving sample nucleic acids to yield cleaved nucleic acids; treatingthe cleaved nucleic acids using a nuclease that preferentially digestsdouble stranded nucleic acid relative to single stranded nucleic acid toyield treated sample nucleic acids; annealing an oligonucleotide to thetreated sample nucleic acids, the oligonucleotide (also referred to asthe “SSP oligonucleotide”) having a promoter region and a target bindingregion that binds to a first target site; and transcribing the annealedtreated sample nucleic acid using an RNA polymerase that recognizes thepromoter region to generate RNA replicates of the sample nucleic acid.The SSP oligonucleotide can include an element that spaces the promoterfrom the insoluble support, e.g., by a distance described herein. Themethod is useful for amplifying sample nucleic acid.

[0039] In one embodiment, the method further includes, prior to orconcurrent with the transcribing, extending the annealed oligonucleotideand/or the annealed sample nucleic acid using a DNA polymerase. The DNApolymerase can lack 3′ to 5′ exonucleases activity. For example, the DNApolymerase can be the Klenow fragment of E. coli DNA polymerase I, or amodified or unmodified bacteriophage DNA polymerase such as SEQUENASE™.In one embodiment, the method includes separating the extended strandsfrom the unannealed and/or unextended sample nucleic acid strands priorto transcription. In another embodiment, only the annealed samplenucleic acid is extended, i.e., thereby rendering the promoter regiondouble stranded and functional. The SSP oligonucleotide can have a 3′modification that prevents its extension.

[0040] The promoter region and the target binding region of the SSPoligonucleotide are described herein below.

[0041] In one embodiment, the SSP oligonucleotide includes a moiety thatis attachable to receiving agent. The receiving agent can be attached toan insoluble support, e.g., a bead or planar surface. In one embodiment,the moiety and receiving agent are members of a specific binding pair,e.g., biotin and avidin (or streptavidin), sugar and lectin, and soforth. In another embodiment, the moiety and receiving agent arechemically reactive with each other. For example, the moiety can be anamino group and the receiving agent can be an activated group thatincludes an electron-withdrawing group on an N-substituted sulfonamide.

[0042] The method can be performed at temperatures of less than about50, 45, or 40° C. In other words, in some implementations, the reactiontemperature never exceeds these temperatures. The method can beperformed under isothermal or substantially isothermal conditions.Further enzymes used in one or more reactions can be added and removedby flowing or otherwise altering the medium that contacts the insolublesupport. In one embodiment, pins or other devices that include the SSPoligonucleotide immobilized thereto can be moved from one reactionmixture to another.

[0043] The cleaving can include shearing, sonication, or digestion usinga cleaving agent such as an endonuclease, e.g., one or more restrictionendonucleases. The restriction endonucleases can specifically recognizea 4, 5, or 6 base pair site. They can digest DNA to produce recessedends, e.g., 5′ overhangs, or blunt ends. The sample nucleic acid can be,for example, DNA or RNA. In a preferred embodiment, the sample nucleicacid is DNA, e.g., genomic DNA, cDNA, or recombinant DNA.

[0044] The cleaving can generate fragments having an average size ofless than about 2000, 1000, 700, or 500 nucleotides or can generate afragment in a region of interest of less than about 2000, 1000, 700, or500 nucleotides. The method can include inactivating the cleaving agentand/or separating the cleaved nucleic acids from the cleaving agent.

[0045] The nuclease that is used to treat the cleaved nucleic acidpreferentially digests double stranded nucleic acid relative to singlestranded nucleic acid. A preferential digestion as used herein, refersto at least a 50-fold difference in Km for the respective substrates.The nuclease can be highly processive. The nuclease can be anexonuclease, e.g., lambda exonuclease or T7 exonuclease.

[0046] The nuclease can be attached to an insoluble support, e.g., abead, such as a paramagnetic bead. The method can further includeseparating the nuclease from the treated sample nucleic acids. Themethod can include inactivating the nuclease.

[0047] The method can further include reverse transcribing the RNAreplicates and/or treating the RNA replicates using a ribonuclease,e.g., RNaseH. In another embodiment, the method can further includetranslating the RNA replicates. In still another embodiment, the methodcan further include analyzing the RNA replicates or DNA copies thereof.The analysis can include determining the identity of a nucleotide or thesequence of a region. The analysis can indicate whether an allele orpolymorphism is present.

[0048] In another aspect, the invention features a method that includes:providing an insoluble support having a plurality of addresses; at eachof the plurality of addresses, depositing or synthesizing anoligonucleotide that includes a 5′ promoter region and a 3′ targetbinding region that is complementary to a target site; contacting asample of nucleic acid to the insoluble support; for each of theoligonucleotides of the plurality of addresses, permitting the targetbinding region to anneal to its target site in the sample, if present;extending the annealed sample nucleic acid using a DNA polymerase (e.g.,thereby rendering the promoter region of the oligonucleotidedouble-stranded); and transcribing the annealed sample nucleic acidusing an RNA polymerase that recognizes the promoter region. Theoligonucleotide can include an element that spaces the promoter from theinsoluble support, e.g., by a distance described herein.

[0049] In one embodiment, the promoter regions are the same among theoligonucleotides of the plurality of addresses. In another embodiment,the promoter regions are different.

[0050] The method can include, prior to the extending or thetranscribing, separating unannealed sample nucleic acids or separateannealed and unannealed sample nucleic acids (e.g., after extending theannealed oligonucleotides to copy).

[0051] In one embodiment, the oligonucleotide is extended using a DNApolymerase.

[0052] In another embodiment, the insoluble support is positioned in aflow chamber. The RNA polymerase and ribonucleotides are provided to thechamber as transcription products are removed from the chamber.

[0053] In still another aspect, the invention provides a method thatincludes: providing an insoluble support having a plurality ofaddresses, each address including (1) a first nucleic acid segmenthaving (a) a 5′ promoter region and (b) a variable 3′ target bindingregion, and (2) a second nucleic acid segment that binds the 5′ promoterregion; annealing sample nucleic acids to the insoluble support; joiningthe 5′ terminus of the second nucleic acid segment to the 3′ end of theannealed sample nucleic acid; optionally removing unjoined and/orunannealed sample nucleic acids; and transcribing the joined samplenucleic acids using an RNA polymerase that recognizes the 5′ promoterregion. The first nucleic acid segment can include an element thatspaces the promoter from the insoluble support, e.g., by a distancedescribed herein.

[0054] In one embodiment, the first nucleic acid segment and the secondnucleic acid segment are segments of a single nucleic acid strand, e.g.,a hairpin strand. The hairpin can include a modified nucleotide orbackbone position in the hairpin loop. The modification includes amoiety that is attached to the insoluble support. The position of thehairpin can be selected such that the 5′ end of the promoter region isspaced from the insoluble support, e.g., by a distance described herein.

[0055] The joining can be effected by a ligase, e.g., T4 DNA ligase, ora thermostable ligase. A thermostable ligase can be useful for annealingat temperatures above 40° C. in order to increase annealing specificity.

[0056] In one embodiment, the method includes storing or archiving theinsoluble support. The insoluble support can be stored any time afterthe joining of the annealed sample nucleic acid, e.g., prior to thetranscribing, or after the transcribing.

[0057] In another aspect, the invention provides a method of analyzinggenetic polymorphisms. The method includes: for each polymorphism,locating a fragment flanked by restriction enzyme sites and includingthe polymorphism such that the sites are less than about 2000, 1000,700, 500 nucleotides apart; synthesizing a promoter oligonucleotidehaving (a) a 5′ promoter region and (b) a variable 3′ target bindingregion, the variable 3′ target binding region being near or flanking oneof fragment termini; optionally attaching the promoter oligonucleotideto an insoluble support; annealing sample nucleic acid to the promoteroligonucleotides; contacting a DNA polymerase to the annealed samplenucleic acids to extend the annealed sample nucleic acid and render thepromoter double-stranded; and transcribing the extended annealed samplenucleic acid using an RNA polymerase specific for the promoter.

[0058] In another aspect, the invention provides a method of analyzinggenetic polymorphisms. The method includes: for each polymorphism,synthesizing a promoter oligonucleotide on an insoluble support, thepromoter oligonucleotide having (a) a 5′ terminus attached to thesupport; (b) a 5′ promoter region and (c) a variable 3′ target bindingregion, the variable 3′ target binding region being within 1000nucleotides (e.g., less than 800, 700, 500, or 400 nucleotides) of thepolymorphism; annealing sample nucleic acid to the promoteroligonucleotides; contacting a DNA polymerase to the annealed samplenucleic acids to extend the annealed sample nucleic acid and render thepromoter double-stranded; and transcribing the extended annealed samplenucleic acid using an RNA polymerase specific for the promoter. Thepromoter oligonucleotide can include an element that spaces the promoterfrom the insoluble support, e.g., by a distance described herein.

[0059] In another aspect, the invention features a method of amplifyinga nucleic acid strand. The method includes: annealing a nucleic acidstrand to a first oligonucleotide that binds to the strand; extendingthe strand 3′ end to form a first oligonucleotide-strand complex;transcribing the first oligonucleotide-strand complex using a first RNApolymerase to yield a first RNA strand; annealing the first RNA to asecond oligonucleotide that binds to the first RNA strand; reversetranscribing the first RNA to yield to a first copy strand; renderingthe first copy strand double-stranded to form a secondoligonucleotide-copy strand complex or annealing a third oligonucleotidethat is complementary to the promoter region of the secondoligonucleotide; and transcribing the second oligonucleotide-copy strandcomplex.

[0060] The first oligonucleotide includes a promoter region,specifically recognized by a first RNA polymerase, and a target bindingregion that binds the strand 3′ end. The second oligonucleotide includesa promoter region, specifically recognized by a second RNA polymerase,and a target binding region that binds the first RNA strand 3′ end. Thefirst and second oligonucleotides can bind to their targets near thetarget 3′ end, e.g., at a location with the strand terminus, or locatednear the strand terminus within 25% of the length of the strand. Thefirst and/or second oligonucleotide can include a spacer, e.g., thatseparates the promoter and the support attachment site by a distancedescribed herein.

[0061] The method can be performed in a homogenous reaction mixture.

[0062] In another aspect, the invention features a kit that includes:(1) a prokaryotic RNA polymerase; (2) a DNA polymerase that lacks 3′ to5′ exonuclease activity; and (3) an exonuclease that is processive andthat preferentially digests double stranded nucleic acid relative tosingle stranded nucleic acid.

[0063] The kit can further include: a promoter oligonucleotide thatincludes (a) a 5′ promoter region that is recognized by the prokaryoticRNA polymerase and (b) a variable 3′ target binding region. In anotherembodiment, the kit includes a plurality of promoter oligonucleotides.In another embodiment, the kit includes an insoluble support that isattached to the promoter oligonucleotide or promoter oligonucleotides.The promoter oligonucleotides can include an element that spaces the 5′end of the promoter form the support by a distance described herein.

[0064] In another embodiment, the kit further includes ribonucleotidesand/or deoxy-ribonucleotides. In yet another embodiment, the kit furtherincludes a container that includes a plurality of restrictionendonucleases. The kit can further one or more reaction containers,e.g., microtiter plates, strips, wells, cassettes, and microfluidicdevices.

[0065] In another aspect, the invention features a pool of non-naturallyoccurring RNA strands.

[0066] The RNA strands are less than about 1000, 700, or 500 nucleotidesin length. In one embodiment, at least some or all of the RNA strandshave a nucleic acid sequence which is absent from fully processed mRNA.For example, the RNA strands can be transcribed from fragments ofgenomic DNA which include introns and/or regulatory regions, e.g.,transcriptional regulatory regions. The RNA strands can include a common5′ end, e.g., corresponding to a linker sequence from an SSPoligonucleotide. The common 5′ end can be about 2 to 50 nucleotides inlength. The 5′ end can include an internal ribosome entry site, aninitiator methionine, and so forth. The RNA can be uncapped.

[0067] In still another aspect, the invention features a reactionmixture that includes: (1) a prokaryotic RNA polymerase; and (2) aplurality of oligonucleotides, each oligonucleotide including (a) a 5′promoter region that is recognized by the prokaryotic RNA polymerase and(b) a variable 3′ target binding region. The mixture can furtherinclude: (3) ribonucleotides. In another embodiment, the mixture furtherincludes: (4) a DNA polymerase that lacks 3′ to 5′ exonuclease activity;and (5) deoxyribonucleotides. In one embodiment, the mixture can be usedto support a homogeneous reaction in which DNA and RNA are synthesized.

[0068] The reaction mixture can further include a second RNA polymeraseand a second plurality of oligonucleotides, each of the oligonucleotidesincluding (a) 5′ promoter region that recognized by the second RNApolymerase, and (b) a variable 3′ target binding region.

[0069] In one embodiment, the target binding region of theoligonucleotides of the second plurality can bind to a strandcomplementary to that bound the target binding region of anoligonucleotide of the first plurality. The two respective targetbinding regions can be within about 4, 2, 1, 0.7, 0.5, 0.3, or 0.1 kb ofone another.

[0070] In still another aspect, the invention features an insolublesupport that includes a plurality of addresses, each address of theplurality having attached thereto an oligonucleotide that has (a) a 5′promoter region that is recognized by a prokaryotic RNA polymerase and(b) a variable 3′ target binding region. The variable target bindingregion can be between about 12 and 50 nucleotides in length. The targetbinding region can have a T_(m) for annealing to its target of betweenabout 24° C. to 85° C., e.g., about 38° C. to 70° C. The insolublesupport can be a bead, a matrix, or a planar surface such as a glassslide, membrane, plastic, or a pliable sheet.

[0071] In still another aspect, the invention features an insolublesupport that includes a first and second plurality of addresses, eachaddress of the first and second plurality having attached thereto anoligonucleotide that has (a) a 5′ promoter region that is recognized bya prokaryotic RNA polymerase and (b) a variable 3′ target bindingregion. At each of address of the first plurality, the promoter regionof the attached oligonucleotide is recognized by a first RNA polymerase.At each address of the second plurality, the promoter region of theattached oligonucleotide is recognized by a second RNA polymerase.

[0072] In one embodiment, the target binding regions of each of theoligonucleotides of the first plurality binds a target site which is ona strand complementary to the target site bound by a target bindingregion of an oligonucleotide of the second plurality.

[0073] The invention also features methods of using the insolublesupport, e.g., the SP-TCR method.

[0074] The invention also features a kit including a first insolublesupport and a second insoluble support. The first insoluble support isan array of SSP oligonucleotides. The second insoluble support is anarray of detection probes, each probe querying an allele of a fragmentamplifiable by the SSP oligonucleotide array.

[0075] In another aspect, the invention features a system that includes:a processor; an array synthesizer; and a repository of polymorphisminformation. The processor is interfaced with the array synthesizer. Thearray synthesizer is receives input information that is used toconstruct an array having 5′ anchored SSP oligonucleotides at eachaddress of a plurality of array addresses. The processor can beconfigured with software to receive a set of polymorphisms for analysis;lookup or compute an appropriate SSP oligonucleotides; and sendinstructions to the array synthesizer to synthesize an array havingprimers for the SSAT amplification of the set polymorphisms or an arrayof detection primers.

[0076] The system can further include an array scanner that is alsointerfaced with the processor. The array scanner can send results fromscanning detection arrays to the processor. The results can be stored ina repository of results.

[0077] In yet another aspect, the invention features a method thatincludes: providing an insoluble support having attachedoligonucleotides; annealing a sample that comprises RNAs to theinsoluble support; extending the attached oligonucleotides using anRNA-directed DNA polymerase to construct DNA replicates of the RNAs;synthesizing DNA strands complementary to the DNA replicates; andtranscribing the complementary strand using an RNA polymerase thatrecognizes the promoter region to produce RNA replicates. Typically, theRNA replicates are anti-sense with respect to the sample RNAs. Thesample RNAs can include RNAs, e.g., obtained from a tissue sample suchas a mammalian tissue sample. The sample RNAs can be obtained from lessthan about 1000, 100, or 10 cells. For example, the sample RNAs can beobtained from about 1, 2, 3, or 5 cells. The mRNA can be is less than 10ng. In one example, the tissue is a normal tissue. In another example,the tissue is tumorous or metastatic.

[0078] The method can further include storing the insoluble support forat least 12, 24, 48, 100, or 200 hours prior to the transcribing, e.g.,and in some cases at least 6 months, or at least a year.

[0079] In one embodiment, the attached oligonucleotides are the same. Atleast some of the attached oligonucleotides can include a T7 promoter, ahomopolymeric T tract, and a terminal A, G, or C. In one embodiment, theattached oligonucleotides are covalently attached to the insolublesupport, e.g., by their 5′ end. In another, they are non-covalentlyattached.

[0080] The RNA replicates can be labeled. The method can further includehybridizing the labeled RNA replicates to a target, e.g., a filter, anucleic acid array, or a solution comprising target nucleic acids.

[0081] The insoluble support can be a surface of a well of a multiwellplate. The insoluble support can be at least partially composed of glassor a plastic.

[0082] In one embodiment, the method further includes hybridizing alabeled probe to the insoluble support.

[0083] In another aspect, the invention features a method that includes:providing an insoluble support having attached oligonucleotides;annealing a sample that comprises RNAs to the insoluble support;extending the attached oligonucleotides using an RNA-directed DNApolymerase to construct DNA replicates of the RNAs; synthesizing DNAstrands complementary to the DNA replicates; ligating an adaptor to theDNA replicates, and transcribing the complementary strand using an RNApolymerase that recognizes the promoter region to produce RNAreplicates. The adaptor can include a promoter region for a second RNApolymerase. The adaptor can further include a unique restriction enzymerecognition site, a translational control sequence, or a sequenceencoding a purification tag.

[0084] The method can further include reverse transcribing the RNAreplicates to form second DNA replicates and transcribing the second DNAreplicates using the second RNA polymerase.

[0085] In still another aspect, the invention features a method thatincludes: providing an insoluble support having a 5′ attachedoligonucleotides; annealing a sample that comprises RNAs to theinsoluble support; and extending the attached oligonucleotides using anRNA-directed DNA polymerase to construct DNA replicates of the RNAs. Inparticular, the invention features an insoluble support made by a methoddescribed herein, such as one of the afore-mentioned methods.

[0086] The invention also features a kit that includes an array of senseprobes and an array of anti-sense probes, wherein for each of at least10, 20, 30, 40, 60, or 80% of the probes on the array of sense probes, acorresponding and complementary probe is present on the array ofanti-sense probes.

[0087] In another aspect, the invention features a method that includes:providing a nucleic acid sample; preparing a first and second populationof single-stranded nucleic acid strands, wherein the strands of thefirst population are complementary to the strands of the secondpopulation; and evaluating the abundance of a plurality of species inthe first population using first probes and the abundance of a pluralityof species in the second population using second probes, wherein thefirst and second probes are substantially complementary. The strands canbe RNA or DNA. In one embodiment, the first probes are attached to afirst planar array and the second probes are attached to a second planararray. The method can further include determining a score that is afunction of the hybridization level of a given sequence to acorresponding first probe and the hybridization level of a complement ofthe given sequence to a corresponding second probe. For example, thescore can be a function of a ratio of the hybridization levels. Themethod can further include repeating the method for a second sample andcomparing the ratio associated with a given sequence between the firstand second sample to the ratio associated with a complement of the givensequence between the first and second sample.

[0088] In another aspect, the invention features a method that includes:assessing transcript levels using sense copies of a pool of transcriptsand anti-sense copies of the pool of transcripts. The method can furtherinclude comparing transcript level detected from the sense copies andantisense copies for a plurality of genes. The comparing can includeevaluating a ratio between the detected transcript levels for differentgenes.

[0089] The methods described herein can produce a population of relevantsingle stranded nucleic acids. The nucleic acids can, for example, allhave the same strandedness. In many embodiments, the product nucleicacid is RNA, which can be enzymatically distinguished from input DNA.Thus, any remnant input DNA can be specifically removed by digestion.Moreover, the methods are particularly suited for multiplex analysis,and, thus, adaptable for applications such as the high-throughputanalysis of multiple nucleic acid polymorphisms. The challenges ofmultiplex analysis are described, for example, in Pastinen et al.((2000) Genome Research 10:1031-1042).

[0090] Further, as many embodiments of the invention do not require PCRor another thermal cycling reaction, many and sometimes all steps can beconducted under isothermal conditions, typically at temperatures such as4° C., 16° C., 25° C., 37° C. or 42° C. Reactions can go for varioustimes, e.g., at least 1, 2, 4, 6, or 12 hours.

[0091] Still another advantage is that the methods are readily adaptedto amplify DNA rather than RNA. In particular, genomic DNA or cDNA canbe analyzed, for example, for polymorphisms. cDNA can be obtained from asingle cell, or from a small number of cells (e.g., less than 10⁶, 10⁵,or 1000, 100, or 50 cells).

[0092] The invention also provides insoluble supports that areeffectively “promoter primer chips.” These chips can be produced inquantity and used to query a relevant subset of a genome. Further, asset forth below, once primed with sample nucleic acids or ligated tosample nucleic acids, the chips can be stored, thereby archiving thesample. Later, the stored chips can be used for additional nucleic acidproduction.

[0093] The chips and other insoluble supports also advantageouslyconcentrate relevant target nucleic acids from a complex sample. Theremoval of non-relevant nucleic acids from a complex sample, beforeinitiating amplification, can further reduce the likelihood ofbackground signals. A background element which appears early in anamplification cycle can dominate species of interest. Some RNApolymerases, such as T7 RNA polymerase, can produce >600 copies of eachtemplate in one transcription reaction. Therefore, two to three cyclesof transcription-based amplification can achieve very high yields. Asexemplified below, the method is highly sensitive. For example, aspecific nucleic acid fragment can be amplified from 100 ng of humangenomic DNA or from a cDNA, e.g., from a single cell.

[0094] A further advantage is that the method enables the production andarchiving of a reproducible nucleic acid library without the use ofcells. The library can be stored, e.g., as an immobilized population ofnucleic acids. Because the nucleic acids are not introduced into cells,representation of nucleic acids in the library is not subjected tobiases that can be caused by cellular toxicity and other unpredictablefactors.

[0095] In addition, as described for some methods, use of an insolublesupport (such as a pin or an array) enables simple exchange of reactionsolutions. For example, enzymes can be removed without complex stepssuch as heat inactivation, phenol extraction, or ethanol precipitation.

[0096] With respect to many embodiments, it is also found thattranscription-based amplification can include designing the promoterposition to create a defined terminus for each RNA product. Probes foreach product are similarly designed.

[0097] An oligonucleotide refers to a nucleic acid of less than 150nucleotides. Oligonucleotides can be produced synthetically orenzymatically (e.g., by excision from a larger nucleic acid). Anoligonucleotide can include a double-stranded region, e.g., byself-annealing or by annealing to another nucleic acid. Anoligonucleotide is typically a DNA molecule, e.g., with an extendable 3′end. An oligonucleotide can include one or more modification (e.g.,attached ligands).

[0098] The details of a number of embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0099]FIG. 1 is a flow chart of the steps in an exemplary SSAT method.

[0100]FIG. 2 is a schematic of the steps in an exemplary SSAT method.

[0101]FIG. 3 is a schematic of an implementation of the SSAT method fora complex input sample and target specific SSP oligonucleotides.

[0102]FIG. 4 is a flow chart of modules that are implemented by anexemplary system.

[0103]FIG. 5 is a schematic of an exemplary system.

[0104]FIG. 6 is a schematic of an example of dual promoter SP-TCRmethod.

[0105]FIG. 7 is a schematic of an example of single promoter SP-TCRmethod.

[0106]FIG. 8 is an example of SNP detection method.

[0107]FIG. 9 is a schematic of an example of attachment by ligation.

[0108]FIG. 10 is a schematic of an example of cycles of TCR.

[0109]FIG. 11 is an exemplary transcription method for producing aRNAand/or sRNA.

[0110]FIG. 12 is an exemplary method for covalently coupling anoligonucleotide to an insoluble support.

[0111]FIG. 13 is an exemplary method for evaluating the coupling of anoligonucleotide to an insoluble support.

[0112]FIG. 14 is an exemplary method for synthesizing a second DNAstrand.

DETAILED DESCRIPTION

[0113] One exemplary application enables the amplification of nucleicacid populations (e.g., mRNA or DNA populations) by transcription usingimmobilized template nucleic acids. An exemplary process is as follows:

[0114] First, an insoluble support is provided. The insoluble supporthas immobilized oligonucleotides that include a promoter sequence and atarget annealing sequence. The 3′ terminus is available for extension bya polymerase. The 5′ terminus of the promoter sequence is spaced fromthe insoluble support by a distance of at least 4 nm. See below “SpacerLengths”. A linker sequence can be present between the promoter sequenceand the target annealing sequence. The linker sequence can include, forexample, one or more of a restriction site (e.g., a 4-, 6- or an 8-basecutter such as AscI), a sequence encoding a purification tag (such asthe hexa-His tag or S-tag), a splicing sequence, and a translationalcontrol signal (such as the Kozak consensus sequence or other ribosomeentry site) or complements thereof.

[0115] For an embodiment in which mRNA is amplified, for example, thetarget annealing sequence can include poly-dT. In some embodiments, thetarget annealing sequence includes a single A, G, or C nucleotide at its3′ terminus. The A, G, or C nucleotide serves to anchor the poly-dTprimer at the 5′ end of the poly-A tract of mRNA. Typically, theinsoluble support includes a population of immobilized oligonucleotideswith different terminal nucleotides, e.g., so that all mRNAs canhybridize to the target annealing sequence. Dinucleotide anchors canalso be used, as can gene or family specific primers. Other targetannealing sequences can also be sued (e.g., target specific nucleic acidsequences or repetitive sequences, e.g., as for some genome nucleic acidsequences)

[0116] The insoluble support is then washed and equilibrated in 1×firststrand synthesis buffer (e.g., 1×first strand synthesis buffer (50 mMTris-HCL, pH 8.3 at 42° C.; 50 mM KCl; 10 mM MgCl₂; 0.5 mM spermidine;10 mM DTT). The mRNA sample is annealed to the immobilizedoligonucleotides in the presence of first strand synthesis buffer (e.g.,including DNase inhibitor). The annealing can proceed at 42° C. for atleast 5 minutes.

[0117] After annealing, cDNA synthesis is initiated by the addition ofsodium pyrophosphate, AMV reverse transcriptase (e.g., from UniversalRiboclone cDNA Synthesis System Catalog No. C4360 from Promega Corp,Madison, Wis., USA) and deoxynucleotides (e.g., 1 mM each of dATP, dCTP,dGTP, dTTP). The reaction can proceed, e.g., at 42° C. for at least 30minutes.

[0118] After synthesis of the first cDNA strand, the insoluble supportnow has attached cDNA copies of each annealed mRNA. The cDNA copies areimmobilized and are operably linked to a promoter since they areconstructed by extension of the immobilized oligonucleotide. Thus, theinsoluble support can be stored at this stage, and then retrieved forlater amplification and analysis.

[0119] A variety of methods can be used to produce the second cDNAstrand, if required. See below (“Second Stand Synthesis”). The insolublesupport can be stored at this point. Typically, the insoluble support iswashed extensively and incubated in a cryoprotectant (e.g., 10%glycerol) prior to storage.

[0120] After second cDNA strand synthesis, in some embodiments, a DNAadaptor is ligated to the free terminus of the immobilized cDNA. The DNAadaptor can include a transcription promoter, e.g., the T3 DNApolymerase promoter. This design is useful for the transcription chainreaction described herein. The adaptor can also include one or more of arestriction site (e.g., a 4-, 6- or an 8-base cutter such as AscI), asequence encoding a purification tag (such as the hexa-His tag orS-tag), a splicing sequence, and a translational control signal (such asthe Kozak consensus sequence or other ribosome entry site) orcomplements thereof.

[0121] The support can be used to generate RNA copies of the originalsample. The support is first equilibrated in RNA polymerasetranscription buffer and then contacted with RNA polymerasetranscription reagents, e.g., T7 RNA polymerase and ribonucleotides(e.g., as provided by AMPLISCRIBE™ T7 High Yield Transcription Kit,Catalog No. AS2607, Epicentre, Madison, Wis.). Reactions areappropriately incubated, e.g., at 37° C. for at least one hour. Afterincubation, amplified mRNA can be harvested from the reaction solution.

[0122] For transcription chain reaction embodiments, the amplified mRNAcan be amplified using the other promoter (e.g., the T3 DNA polymerasepromoter) as described above.

[0123] The insoluble support serves as a DNA archive of the originalmRNA sample. The archive can be returned to, time and again. Moreover,the archive is amplified by transcription, which restores the originalsample in its RNA state. Such amplification is also linear and may beless susceptible to biasing events than, e.g., exponentialamplification. In some embodiments, the method is supported by a singleprimer for reverse transcription. The primer is universal for allpolyadenylated mRNAs.

[0124] Spacer Lengths

[0125] We have discovered that nucleic acid amplification according tothe methods described herein is remarkably improved by spacing thepromoter sequence from the insoluble support. The distance between the5′ terminus of the promoter and the support is at least 4 nm, and isdescribed with additional particularity as follows and by Examples 1-4,below:

[0126] 1) Non-Covalent Immobilization:

[0127] Immobilization oligonucleotides can be immobilized bynon-covalent interaction between a ligand that is covalently attached tothe oligonucleotide and a protein immobilized on the support. Forexample, the ligand can be biotin and the interaction can be betweenbiotin and a biotin-interacting protein (e.g., streptavidin or avidin).When this configuration (ligand/ligand-binding protein, e.g.,biotin/biotin-interacting protein) is used to immobilize theoligonucleotide to the insoluble support, optimal spacings between theimmobilized nucleotide position (e.g., the 5′ nucleotide) and the 5′ endof the promoter sequence is at least 5 nucleotides, e.g., between 6 and20 nucleotides, or between 6 and 15, or 6 and 12 nucleotides, e.g., 6,7, 8, 9, 10, 11, or 12 nucleotides. In one embodiment, it is alsopossible to substitute the nucleic acid spacer sequence with anon-nucleic acid spacer, e.g., a chemical linker described below.

[0128] Other examples ligand/ligand-binding protein interactionsinclude: FK506 and FK506BP, chitin and chitin binding protein; celluloseand Cellulase (CBD); amylose or maltose and maltose binding protein;methotrexate and dihydrofolate reductases.

[0129] 2) Covalent Immobilization

[0130] Oligonucleotides can be immobilized by covalent couplingchemistries, e.g., by a homopolymeric linker (e.g., a polyethyleneglycol linker), and phosphate linkages. Additional examples ofhomopolymeric linkers include: polymers with subunits having 2, 3, or 4main chain atoms, and between 5 and 12 repeats of the subunits. Forexample, the linker can be composed of polyethylene glycol((CH₂CH₂O)_(n)—, e.g., where n is >8, e.g., 8 to 20, 8 to 16 or 8 to 12)and/or polymethylene, ((CH₂)_(n)—, e.g., where n is >18, e.g., 18 to 60,or 24 to 48). Generally it is possible to use any chemical linker (e.g.,without nucleotide units) that has the same physical length or atomlength as a linker described herein, e.g., a homopolymeric linkerdescribed above, e.g., including 18 to 60, or 24 to 48 main chain atoms.

[0131] An oligonucleotide that includes a nucleotide that is directlycoupled to the immobilized support can include a spacer of between 15and 45 nucleotides, e.g., between 20 and 35 nucleotides, e.g., between23 and 28 nucleotides, or about 25 nucleotides. The oligonucleotide canbe coupled, e.g., using carbodiimide activation.

[0132] In one embodiment that uses carbodiimide activation, a primeroligonucleotide containing a 5′-phosphate group is activated with ethyl1,1-dimethylaminopropylcarbodiimide hydrochloride (EDC) in the presenceof 1-methylimidazole. See, e.g., Chu et al. (Nucleic Res., 11, 6513(1983). The active phosphate ester intermediate is converted into areactive phosphorimidazolide which reacts spontaneously with an aminogroup on the insoluble support. This covalent coupling chemistry furtherensures that only the 5′-end of the primer oligonucleotide is attachedto the insoluble support. See, e.g., FIG. 12.

[0133] Other attachment chemistries include: coupling between anamine-including oligonucleotide and an activated carboxylate group orsuccinimidyl ester; coupling between a thiol-including oligonucleotide(SH-oligo) and an alkylating reagent such as an iodoacetamide ormaleimide; coupling of an Acrydite-oligonucleotide through a thioether.See, e.g., Adessi et al. (2000) Nucleic Acids Research 28:e87; Ghosh,and Musso (1987) Nucleic Acids Res. 15:5353-5372; Lindroos et al. (2001)Nucleic Acids Res. 29:e69; Rogers et al. (1999) Anal. Biochem.266:23-30.

[0134] Nucleotide regions of spacers can be prepared using any nucleicacid sequence, for example, a homopolymeric sequence, a low complexitysequence, a medium complexity sequence, a complex sequence, or asequence absent from the sample of relevance. A “low complexitysequence” is a sequence that includes repeating units of 4, 3, or 2nucleotides. A “medium complexity sequence” includes fewer than threetypes of repeats, each repeat being 2, 3, 4, or 5 nucleotides. Asequence absent from a sample of relevance can be identified bysearching a computer database of sequences potentially in the sample.For example, if the sample is human, it is possible to identify a spacerthat is not present in human genome sequence or that is notcomplementary to any human genome sequence.

[0135] Second Strand Synthesis

[0136] One method includes reaction with a strand-displacing DNApolymerase (e.g., DNA polymerase I) and RNase H, e.g., at 16° C. RNase His used to nick the RNA strand. If necessary, the reaction can becompleted by T4 DNA polymerase. The second cDNA strand formshomoduplexes of DNA on the array and thereby contributes to stability.

[0137] Another method includes tailing of the extended immobilizedoligonucleotide by terminal transferase, e.g., in the presence of dGTPso that a polyG tail is add, e.g., about 10-20, 12-18, or an average of15 nucleotides in length. After the tailing, a primer that includes C isannealed, and optionally ligated, and extended. See FIG. 14. Seegenerally, e.g., Okayama and Berg Mol. Cell. Bio. 2:161-170, 1982;Spickofsky and Margolskee Nucleic Acids Res. 19: 7105-7111, 1991);Dugaiczyk, et. al. Biochemistry 19:5869-5873, 1980).

[0138] It is also possible for a hairpin to form spontaneously such thatthe terminal nucleotide can be extended to form the complementarystrand.

[0139] Still another method uses primer corresponding to the 5′ end ofthe target sequence, e.g., complementary to the 3′ end of the extendedoligonucleotide. Random hexanucleotides or other priming sequences canalso be used, particularly after removal of the RNA strand, e.g., bydenaturation or nicking with RNase H.

[0140] SSP Oligonucleotide Design

[0141] SSP oligonucleotides are used to attach a promoter for an RNApolymerase to a DNA template. The SSP oligonucleotides used in thismethod generally have a length of 25 to 100 nucleotides, e.g., about 30to 50 or 40 to 60 nucleotides. The SSP oligonucleotides are alsodesigned so that the promoter sequence is spaced from the insolublesupport when the SSP oligonucleotide is immobilized. The spacing can beas described above.

[0142] An SSP oligonucleotide has a 3′ sequence, also termed “targetbinding region,” which anneals to a target site within a targetfragment. This sequence can be substantially homologous, e.g., 90 to100% identical, to the target site. An identical or nearly identicalsequence increases specificity of amplification. The length of thetarget binding region can be selected such that the T_(m) for a duplexformed between it and the target site is at least about 42° C., 50° C.,or 55° C. The target binding region can be optimized such that it doesnot anneal to itself or the remainder of the SSP oligonucleotide (e.g.,form hairpins).

[0143] An SSP oligonucleotide also contains a promoter sequence 5′ endto the target sequence. The promoter sequence is recognized by an RNApolymerase. The RNA polymerase can be prokaryotic, eukaryotic, orarcheal. For example, the RNA polymerase can be a prokaryoticbacteriophage RNA polymerase such as the T7, T3, and SP6 RNApolymerases. Hence, exemplary promoter sequences include, but notlimited to, T7, T3, Sp6 RNA polymerase promoters sequences. Generally,any RNA polymerase that can be specifically directed to a promoter canbe used. For example, SPO promoters can be used in conjunction withsigma factors from the Bacillus subtilis phage SP01 to target RNApolymerase to SP01 promoters.

[0144] The SSP oligonucleotide can be attached or attachable to aninsoluble support. For example, the SSP oligonucleotide can also includea modification to facilitate affinity capture of the target or of theduplex formed by extension of the target-SSP oligonucleotide complex.The modification can include, for example, a small molecule of less than1000 Daltons molecular weight for binding by a protein that binds (e.g.,specifically binds) to the small molecule. For example, one or morebiotinylated deoxynucleotides (or other ligands) can be used. Otheruseful modifications include amino and thiol moieties. A biotinylatedmoiety can be bound to immobilized streptavidin or avidin. Other usefulnon-covalent and covalent linkages are widely known. For some reactions,e.g., using a biotinylated SSP oligonucleotide, about 0.1, 1, 5, 10, 20,or 100 pmol of SSP oligonucleotide are used per reaction. The reactionmight be about the size of a well of a 96-well carrier.

[0145] In one embodiment, the primer includes a sequence have one strandof a restriction enzyme recognition site. The primer can also include amodified base, such as α S-dNTP, within the recognition site, e.g., suchthat the primer strand is not cleaved. DNA polymerase will thenrecognize the nick, and start polymerization which results indisplacement of the nicked DNA strand. Repeat nicking and polymerizationlead to linear amplification of one strand of the target DNA.

[0146] Optionally, a linker sequence can be included between the SSPoligonucleotide promoter sequence and the target sequence. The linkersequence is transcribed, and can include restriction endonuclease sites(e.g., sites for a 6- or 8-base pair cutter) to facilitate cloning ofthe amplified nucleic acids, a synthetic identification tag, or auniversal sequence. The linker region can include a sequence that isrecognized by an RNA binding protein when the linker region istranscribed into RNA. Exemplary RNA binding proteins include Tat andNus.

[0147] In one embodiment, the linker region includes an internalribosome entry site, an initiator methionine, an epitope tag, apurification or detection tag, and/or a translational regulatorysequence.

[0148] Further exemplary methods of SSP oligonucleotide design are alsodescribed in “Ligation” and “Software,” see below.

[0149] Once designed SSP oligonucleotides can be synthesized usingstandard oligonucleotide synthesis chemistry. Further, if a clone bankis generated, SSP oligonucleotides can be produced enzymatically (e.g.,by PCR) or by isolation from a host cell (e.g., E. coli).

[0150] SSP Oligonucleotide Annealing

[0151] The SSP oligonucleotides are annealed to single stranded DNA fromthe exonuclease treatment. The annealing can be performed at atemperature below the T_(m) of the SSP oligonucleotide for its targetbinding site. Hybridization of SSP oligonucleotides to the singlestranded target fragments can be performed in any container, e.g., atube, such as a micro-centrifuge tube, a well, or a flow cell. The SSPoligonucleotide can be attached to an insoluble support, either before,during, or after annealing.

[0152] A variety of hybridization conditions can be used. Hybridizationconditions are described, for example, in standard laboratory manualssuch as (Molecular Cloning, 3^(rd) edition, Cold Spring Harbor Press,ed. Sambrook & Russell). Temperature and salt concentration can beselected to achieve the desired stringency.

[0153] One method is to hybridize the single-stranded targets to 5′→3′directionally anchored SSP oligonucleotides as is illustrated in FIG. 3.After hybridization, unbound DNA can be removed by washing with buffers.

[0154] Template Extension

[0155] DNA polymerase is used to append the SSP oligonucleotide to thetarget sequence by primer extension, thereby forming double-strandedDNA. Exemplary DNA polymerases include the Klenow fragment (3′-5′ exo⁻),and SEQUENASE™ 2.0 (Amersham Pharmacia Biotech). Any DNA polymerase maysuffice, particularly those lacking 3′ to 5′ exonuclease activity.Conditions for double-stranded DNA synthesis are described, for example,in Gubler (1987) Methods Enzymol 152: 330-335.

[0156] The DNA polymerase can extend the annealed target nucleic acidsegment using the promoter (or other non-target binding region) of theSSP oligonucleotide as a template. This step renders the promoter regiondouble-stranded and functional. Further the extension process “operablylinks” the promoter to the target fragment. As used herein, the term“operably linked” refers to a functional linkage between the affectingsequence (typically a promoter) and the controlled sequence.

[0157] Since a double stranded region is optional in the region afterthe +1 site of the promoter, at least for the bacteriophage RNApolymerases such as SP6, T7, and T3, in some embodiments the 3′terminals of the SSP oligonucleotide can be blocked. In theseimplementations only the promoter region is rendered double-stranded.The SSP oligonucleotide is not used as a primer, but as a template.

[0158] In other embodiments, the SSP oligonucleotide is also extended.This implementation is useful as it renders both the promoter and thetarget double stranded. The extended nucleic acids can be stored as DNAduplexes. Such stored nucleic acid has the advantage of conformationaland chemical stability.

[0159] Ligation

[0160] In another embodiment, depicted in FIG. 9, the target fragment isligated to the bottom strand of an SSP duplex, which includes both theSSP oligonucleotide, and a complementary strand. The three componentstrands can be added in any order. Since the template for transcriptioncan be single stranded (see below), so long as the promoter isdouble-stranded, the asymmetric hybrid formed by the three componentstrands is sufficient for transcriptional amplification. Two componentscan also be used, for example, if the SSP duplex is formed from ahairpin nucleic acid that includes the SSP oligonucleotide sequences andthe complementary region.

[0161] Amplification by Transcription

[0162] The T7 polymerase polypeptide can be isolated from the clonedgene, the T7 gene 1, see e.g., U.S. Pat. No. 5,869,320 (Studier et al.).T7 RNA polymerase can be purified from induced cells that have a nucleicacid for T7 gene operably linked to an inducible promoter. Chamberlin etal., (1970) Nature, 228, 227-231 describes one exemplary scheme forpurifying the polymerase.

[0163] T7 RNA polymerase is highly specificity for its promoter site(Chamberlin et al., in The Enzymes, ed. P. Boyer (Academic Press, NewYork) pp. 87-108 (1982)). The T7 polymerase recognizes a highlyconserved sequence spanning about bp −17 to about +6 relative to thestart of the RNA chain (Dunn and Studier, (1983) J. Mol. Biol. 166:477-535 and (1984) J. Mol. Biol. 175: 111-112. The essential region ofthe promoter extends from −17 to +1. Moreover, the only region of thetemplate strand that must be double-stranded DNA is this region. Theremainder of the template can be single-stranded.

[0164] T7 RNA polymerase is particularly useful for amplification ofdiverse nucleic acid sequences as a result of the dearth of efficienttermination signals for T7 RNA polymerase (see, Rosenberg et al., (1987)Gene 56: 125-135. The T7 RNA polymerase is available, e.g., from PromegaBiotech, (Madison, Wis.) and Epicentre Technologies, (Madison, Wis.).

[0165] SP6 and T3 RNA polymerases have similar properties. Further, eachof these three polymerases is highly specific as it does not transcribenon-cognate promoters. The minimal efficient promoter sequences forthese polymerases are listed in Table 1 below. The +1 nucleotide isunderscored. Other prokaryotic promoters can be used, e.g., a promoterrecognized by an E. coli RNA polymerase. The 5′ end of a promoter can bedefined, e.g., by mutational analysis (e.g., deletion mapping), whereinthe 5′ end bounds the minimal region that is sufficient to provide atleast 70% of the wildtype promoter's activity. TABLE 1 Bacteriophage RNAPolymerase Promoters RNA polymerase Specific Promoter Sequence T7TAATACGACTCACTATAGG (SEQ ID NO:23) T3 AATTAACCCTCACTAAAGG (SEQ ID NO:24)SP6 ATTTAGGTGACACTATAGA (SEQ ID NO:25)

[0166] To obtain amplified RNA, RNA polymerase reaction buffer, excessof all four ribonucleotides, and the corresponding RNA polymerase enzymeare added, and incubate at 37° C. for 2-24 hours. In vitro transcriptionis described, e.g., in Melton, D. et al. (1984) Nucl. Acid. Res.12:7035. The transcription reaction buffer can include a variety ofcomponents, e.g., including:

[0167] 1 to 20 mM NaCl

[0168] 24, 34, or 40 mM Mg₂Cl₂

[0169] 10 to 50 mM Tris·HCl (pH about 7.3, 7.4, or 7.5)

[0170] 1, 2, 3, 5, 7.5, 10 mM rNTP

[0171] 1, 3, 5, 10 mM DTT

[0172] 2 U/μl SuperaseInhibitor

[0173] To obtain labeled RNA to be used as hybridization probe insequence analysis, one or more labeled ribonucleotides are also added.Depending on the intended detection method, the labels can be, but notlimit to, fluorescent dyes such as fluorescein and the cyanine dyes(Cy3, Cy5, Alexa 542, and Bodipy 630/650); radiolabels such as ³² P,³³P, ³⁵S, and ³H; colorimetric or chemiluminescence; and binding paircomponents such as biotin or digoxygenin.

[0174] Post Processing, Archiving, and Storage

[0175] The method can further include any of a number of post-processingsteps. For example, the RNA products can be reverse transcribed into DNAusing specific or random primers. Clearly, the RNA products can be usedfor a variety of purposes. For example, the RNA products (if they havethe appropriate strandedness) can be translated, and the translationproducts analyzed, e.g., for an activity or by contacting thetranslation products with an antibody. Translation products can beanalyzed, e.g., to evaluate one, two, three or more criteria about eachproduct, e.g., using gel electrophoresis, 2D gel electrophoresis, massspectrometry, and other methods. The information can be stored in adatabase, e.g., using a record that includes two, three or more fields,e.g., to provide a multidimensional vector.

[0176] The RNA can be quantitated, e.g., to determine the abundance ofdifferent species. If the RNA is labeled, it can be hybridized to anarray of positional probes for the different known RNA species. In somecases, the RNA is itself functional, e.g., the RNA is an aptamer or acatalyst. Such RNA can be analyzed for binding or catalytic properties.

[0177] Anchored, promoter appended DNA target can also be reused and/orstored for future reference. For example, if a chip of SSPoligonucleotides is used, the chip can washed free of reagents. Thewashed chip can either be immediately reused for additional rounds oftranscriptional amplification or stored, e.g., in an archival process. Astored chip can be dehydrated and frozen, or coated with acryoprotectant such as a glycerol solution, and frozen. When desired, astored chip can be retrieved, washed, and applied with fresh reagentsfor transcription, e.g., ribonucleotides and the appropriate RNApolymerase. As described below, a variety of insoluble supports (e.g.,pins, microtitre wells, spin cups, matrices, and membranes) can be used.

[0178] Likewise, with respect to the cyclic TCR method, in which twodifferent sets of templates (e.g., T7 and T3) are produced, thetemplates may be archived separately or together.

[0179] By coupling templates from different cycles to separate supports,a master and working set of templates can be generated. The working setscan be distributed to different users (e.g., customers). The master setcan be used to produce additional working sets and may also be storedfor reference or quality control.

[0180] Transcription Chain Reaction (TCR)

[0181] Turning now to FIG. 6, increased amplification is achieved byusing the RNA products as templates for additional transcription. TheRNA products are converted to DNA by reverse transcription in a formatanalogous to the process described above for SSP oligonucleotidedirected synthesis. The RNA transcripts made from the SSPoligonucleotide appended double stranded DNA, denoted as (+) targetstrand, are captured by a second SSP oligonucleotide which contains asequence complementary to the newly synthesized RNA strand, e.g., at the3′ end of the RNA strand. The promoter segment of the second SSPoligonucleotide can be different from the promoter of the first SSPoligonucleotide. The captured (+) RNA can now be converted todouble-stranded DNA by reverse transcriptase and DNA polymerase.

[0182] Transcription from these newly synthesized DNA produces RNAcorresponding to the (−) strands of the target, and results in enhancedamplification. The method can be used to detect sequences at very lowconcentrations, e.g., from a single cancer cell in a population ofnormal cells.

[0183] As described above, as the nucleic acid promoter-target fusionsare captured, the insoluble support attached to the first and second SSPoligonucleotides can be stored for later rounds of transcription.

[0184] In one embodiment, the insoluble support contains multiple pairsof first and second SSP oligonucleotides, e.g., to amplify multipledifferent targets.

[0185] Referring to FIG. 7, RNA products from an initial amplificationstage are reverse transcribed, e.g., using a target specific primer. TheDNA strand from reverse transcription can be rendered single stranded,e.g., by mild alkali hydrolysis, heat treatment, 50% formamide at 50°C., or ribonuclease digestion, e.g., using RNaseH. The single strandedDNA replicates can then anneal to the available immobilized SSPoligonucleotides. The process allows for enhanced amplification.

[0186] Referring to FIG. 8, a fragment containing a single nucleotidepolymorphism at a query site is amplified using the SSAT method. Then,RNA products are hybridized to immobilized reverse transcriptionprimers. The reverse transcription primers position their ultimate 3′nucleotide opposite the query site. Reverse transcription only proceedsif the ultimate 3′ nucleotide is complementary to the query sitenucleotide. The incorporation of label, e.g., a Cy3 labeled dNTP can beused to monitor the process.

[0187] Referring to FIG. 10, cycles of T7 (left) and T3 (right)amplification are shown. mRNA or sRNA (from a cycle of T3 amplification)are hybridized to SSP oligonucleotides (e.g., T7-d(T)nV) that areattached to support. The SSP oligonucleotide can include a spacersequence between the promoter and the insoluble support attachment site.For example, the spacer can be at least 6, 12, 18, or 24 nucleotides inlength. Template DNA is produced, e.g., by cDNA synthesis. An adaptormolecule is ligated to the template in an initial cycle (e.g., this isoptional if sRNA is used). The adaptor preferably includes a tagsequence that is absent from the sample. A computer program can be usedto predict sequences that should be absent from a sample obtained from aparticular organism, e.g., by comparison to a comprehensive database ofgenomic or cDNA sequences from that organism. After ligation of theadaptor, transcripts (aRNA) are produced using T7 polymerase.

[0188] The transcripts include the tag sequence as well as the sequencefrom the sample nucleic acid. Transcripts are then hybridized to aT3-TCR SSP oligonucleotide attached to the same support or anotherinsoluble support (see circled A flowchart indicator). Again, cDNA isproduced from the annealed transcripts. The T3 polymerase is now used toproduce sRNA. The sRNA can be cyclic deployed to produce additional aRNAtranscripts (see circled B flowchart indicator).

[0189] Joining of the adaptor can be implemented in a variety of ways.For example, DNA and RNA ligases can be used (e.g., to ligate apreformed duplex that includes the tag sequence). In one embodiment,shown in FIG. 11, terminal transferase and dGTP are used to add ahomopolymeric G tail to the DNA strand. An oligonucleotide that includesthe tag sequence and a 3′ polymeric C tail is annealed and used to primesynthesis of the second DNA strand.

[0190] In some implementations, it is also possible to detect a nucleicacid replicate made from a template by hybridization of a probe that iscomplementary to an adaptor sequence.

[0191] Insoluble Supports

[0192] As described herein, many embodiments include producing templatesfor RNA transcription that are attached to support. The insolublesupport can be composed of any insoluble material. In one example, theinsoluble support is a rigid planar device such as a chip (e.g., amicroscope slide). In another example, the insoluble support is areaction vessel such as a multi-container sample carrier (e.g., amicrotitre plate), tube, column, spin-cup, disposable pipet tip, ring,disc (e.g., paper disc), lantern, pestle, membrane, or portions thereof.For example, the templates can be attached to a surface within one ormore microtitre wells (e.g., in a variety of formats, including single,strips, 96-well, 384-well, robotically manipulated single or multipleplates). The microtitre plates can conveniently be placed intothermocontroller units, e.g., thermocyclers in order to finely controlreaction temperatures.

[0193] In one embodiment, a spin cup is used. The cup has a porousmembrane, e.g., a 0.45 μm membrane or any size membrane that facilitatespassage of macromolecules such as the reaction enzymes. To conduct a setof multiple reactions, the reaction components are passed through themembrane (e.g., by low-speed centrifugation). To switch reactioncomponents, buffer or a subsequent reaction mixture is washed throughthe membrane. The SSP oligonucleotide is first physically attached tothe membrane (e.g., by a non-covalent or covalent linkage). Thus,throughout the reactions the SSP oligonucleotides and templates thatincorporate them remain within the membrane of the spin cup. Aftertemplates are generated, the membranes can also be archived, e.g., forsubsequent RNA transcription. Transcription products can also becollected by low speed centrifugation of the spin cup.

[0194] In another embodiment, a pin or set of pins is used. The SSPoligonucleotides are physically attached to the pins. For example, theSSP oligonucleotides are biotinylated and the pin surface is coated witha streptavidin. To process multiple reactions, multiple pins can berigidly fixed to a holding unit. The holding unit is used to transfer topins to different reaction mixtures. For example, the holding unit canbe a lid of a microtitre plate. The lid is placed on different plates,each plate including appropriate reaction mixtures (e.g., for samplehybridization, reverse transcription, second strand synthesis, andtranscription). For cyclic TCR, alternating pins, each pin for capturingT7 and T3 templates.

[0195] Insoluble supports can be evaluated by hybridizing a probe thatcontains a sequence complementary to the primers on the support. See,for example, FIG. 13. For example, to detect the target binding activityof an immobilized primer, a probe oligonucleotide of5′-biotin-GCGCCAATTATCGAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:27) is allowed tohybridize with the primer on the insoluble support. The hybridizedcomplex is washed with buffer, and then treated with astreptavidin-horse radish peroxidase conjugate. After the reactionbetween streptavidin and biotin molecules are completed on the insolublesupport, the unbound enzyme conjugates are washed away. A solutioncontaining o-phenylenediamine and hydrogen peroxide is then added to theinsoluble support. Color development of the solution is measured toindicate the quantity of streptavidin enzyme conjugate bound to theinsoluble support. This in turn indicates the amount of biotin bound tothe insoluble support which indicates the binding activity of the TCRprimer on the insoluble support.

[0196] Detection Methods

[0197] A variety of detection methods can be used to analyze the RNAproducts of the amplification, or the reverse-transcribed DNA copies ofthe RNA products. Exemplary methods include single base extension (U.S.Pat. No. 6,013,431), mismatch detection (e.g., using MutS protein orother mismatch binding protein), sequencing by hybridization (U.S. Pat.No. 5,202,231 and PCT 89/10977) and RNA sequencing. Pastinen et al.,supra describes an allele specific detection method in which two primersare annealed to the target. The primers differ only at the 3′ most endwhich is complementary to the query site if the primer is directed tothe allele that is present. Labeled nucleotides are only added to theprimer in an extension reaction, e.g., using reverse transcriptase, ifthe primer is complementary at the query site.

[0198] To analyze a profile of sample nucleic acids, labeled RNAproducts can be generated from a template array to replicate the samplenucleic acids. The replicates are hybridized to a detection array thatincludes a plurality of capture probes. The detection array can bescanned to determine whether and to what extent the labeled RNA productshybridize to the probes. Because each probe is at a unique address, theamount of each species can be inferred. Methods for hybridization todetection arrays are well known. The information obtained by analyzingthe detection array can be stored in a machine-accessible medium, e.g.,with a pointer to information about the location or identify of anarchival template array that can be used to make RNA replicates of thesample nucleic acid.

[0199] Paired-Probe Arrays

[0200] One implementation of the invention includes preparing sense andanti-sense nucleic acid from a nucleic acid sample, e.g., from a sampleof mRNA. The mRNA is amplified using an array of immobilized SSPoligonucleotides. A T7 promoter-poly d(T) SSP oligonucleotide hybridizesto the polyadenylated 3′ region of mRNA, dsDNA is synthesized and a TCRadaptor is ligated to the free end. The dsDNA is then transcribed toproduce labeled anti-sense RNA. The anti-sense RNA can also behybridized to an array that includes a TCR-adaptor complementary regionand a RNA polymerase promoter. Labeled sense RNA is produced from thishybridization as described.

[0201] For example, the labeled anti-sense RNA is hybridized to an arrayof sense probes(e.g., an “sOligo Microarray”) and the labeled sense RNAis hybridized to an array of anti-sense probes (e.g., an “aOligoMicroarray”). Data is collected from the two hybridizations andcompared. For example, a transcript ratio is determined for a given genein two different tissue samples using the labeled anti-sense RNA; andanother transcript ratio is determined for the given gene in two tissuesamples using the labeled sense RNA. The two ratios are then compared,e.g., to determine a reliability coefficient (R_(c)) R_(c) valuesbetween 0.8 and 1.0 can be indicative of a reliable observation.

[0202] Other types of ratios can also be determined. For example, for asingle sample, the ratio of levels of hybridization of the labeled senseRNA to probe A and probe B can be compared to the ratio of level ofhybridization of the labeled anti-sense RNA to probe A′ and B′, whereprobe A and A′ are complementary and B and B′ are complementary. Theprobes can be partially or non-overlapping probes to the same gene(e.g., transcript), or can be probes to different genes. In oneembodiment, one of the genes is a housekeeping gene or other gene whoseexpression level provides a useful reference.

[0203] dsRNA

[0204] In one embodiment, double-stranded RNA is produced for one or aplurality of target sequences. The dsRNA can be delivered to cells or toan organism. Endogenous components of the cell or organism can triggerRNA interference (RNAi) which silences expression of genes that includethe target sequence. It is well established that many cells andorganisms have an RNAi response (e.g., nematodes, plant cells, andmammalian cells). Individual target sequences can be annealed to SSPoligonucleotides on different insoluble supports (e.g., differentmicrotitre wells or different pins) in order to generate templates foraRNA and sRNA. These templates can be made separately or on the samesupport. Transcription of these templates produces aRNA and sRNA thathybridize to each other to produce dsRNA. If the aRNA and sRNA areproduced separately, it may be useful to denature the RNAs and annealedthem to form the dsRNA duplex. In some cases, the templates are producedin pools thereby producing a mixed population of dsRNA. In other cases,individual species of dsRNA are produced so that each target sequencecan be separately attacked. The individual species can be correspond todifferent transcripts of an organism or cell, or may correspond todifferent regions within the different transcripts. Some species can besplice variant specific.

[0205] In one embodiment, the templates are immobilized in a regulararray such that each address of the array includes a substantiallyhomogenous population of templates. Cells (e.g., mammalian culturecells) can be grown on the array so that dsRNA made by each address ofthe array can enter the cell. After incubation, the cells can beevaluated to determine the effect of the dsRNA on the cells. The regulararray format can be, e.g., a microtitre plate.

[0206] In one embodiment, dsRNA is made for a substantial portion of atranscriptome. In this case, the plurality of targets is thecorresponding portion of the transcriptome. These dsRNAs can be used,for example, to characterize the biological function of differentmembers of the transcriptome.

[0207] Again, the templates used to produce the dsRNA can be archivedand also produced as master and slave sets. The slave sets can bedistributed to different users who can produce dsRNA on demand. Becausethe template are immobilized, the dsRNAs can be washed from theinsoluble supports and used directly, e.g., contacted to cultured cellsor cell in an organism.

[0208] In one embodiment, a multi-well plate is used. Each well of theplate includes an immobilized SSP oligonucleotide. Different nucleicacids are deposited in each well of the plate and annealed to the SSPoligonucleotide (e.g., by hybridization to a target region of the SSPoligonucleotide) to form an annealed complex. A template is generatedfrom the annealed complex, e.g., using a DNA polymerase (and if theadded nucleic acid is RNA by reverse transcription). A second promoteris joined to the annealed complex or template so that ultimately atemplate is formed with a promoter at both termini. The second promotercan be the same or different from the promoter of the SSPoligonucleotide. RNA polymerase is then added so that transcripts aremade from both strands of the template. The transcripts are annealed toeach to form dsRNAs.

[0209] Exemplary applications for dsRNAs include target validation andtherapeutic use.

[0210] Fragment Preparation

[0211] Referring to FIG. 1, another exemplary implementation of theinvention is set forth. This implementation is directed to the analysisof genomic DNA, e.g., for polymorphisms. The implementation includes:fragment preparation 10, rendering the sample single-stranded 12, SSPoligonucleotide annealing 14, attachment 16, transcription 18, anddetection or analysis 20.

[0212] Genomic DNA is isolated from cells, e.g., from a subject such asa human patient. The DNA is digested using restriction enzymes togenerate target fragments. To amplify multiple fragments from thegenomic DNA, restriction enzymes are selected based on one or more ofthe following criteria. The target fragments are less than about 2000,1000, 500, 700, 500, 300, 200 or 100 nucleotides in length. The targetfragment includes at least about 15, 18, 20, or 22 nucleotides ofnon-polymorphic nucleotide sequence in proximity to the restrictionsite. Such non-polymorphic regions can function as annealing sites forthe SSP oligonucleotide. The polymorphism of interest is located withinthe central two-thirds of the target fragment. If multiple restrictionenzymes are required, the restriction enzymes can be chosen that arecompatible, e.g. functional at the same reaction conditions.

[0213] Referring to the example depicted in FIG. 2, enzyme E cuts theDNA into fragments, labeled “a”, “b”, “c”, and “d”. Fragment “d”contains a sequence of interest that, for example, includes apolymorphism represented by a closed circle.

[0214] In one embodiment, prior to annealing of the SSP oligonucleotideto the sample nucleic acids, the cleaved sample nucleic acids arerendered as single stranded. The production of single-stranded DNA canbe achieved by heat or chemical denaturation. However, enzymatic meansfor producing single-stranded DNA were found to be particularlyeffective for the SSAT method.

[0215] The double-stranded DNA fragments are treated with anexonuclease, such as T7 exonuclease or lambda exonuclease. For example,the cleaved sample nucleic acids can be treated with lambda exonucleasefor about 1 hour at 37° C.

[0216] These exonucleases catalyze digestion of DNA in the 5′ to 3′direction, thereby sequentially removing 5′ mononucleotides from duplexDNA (Little, J W (1981) Gene Amplification and Analysis 2:135-145;Shimozaki and Okazaki. (1978) Nucl. Acids. Res. 5:4245-4261). Thereaction can be inactivated by heating at 75° C. for 30 minutes.

[0217] Lambda exonuclease is a highly processive enzyme. As such, it hasa strong predilection to remain attached to a substrate DNA strand anddigest it to completion before dissociating and attacking anothersubstrate DNA. This feature results in longer single stranded DNAproducts rather than multiple fragments that are a fraction of the sizeof the input DNA. The processivity of various exonucleases is described,e.g., in Thomas and Olivera (1978) J Biol Chem 253:424-9.

[0218] The processed single stranded DNA products are used as samplesfor amplification, e.g., as described above.

[0219] Software

[0220] Also provided is a system and software which can assist, control,and manage one or more steps of the method described herein.

[0221] Referring to FIGS. 4 and 5, software can include modules for oneor more of the following: (1) selecting polymorphisms for analysis 110;(2) identifying restriction enzymes for fragment preparation 120; (3)identifying and, optionally, optimizing SSP oligonucleotide design 130;(4) interfacing with an oligonucleotide synthesizer or oligonucleotidearray synthesizer to produce SSP oligonucleotides 140; (5) synthesizinga detection array 150; and (6) receiving 160 and analyzing 170 resultsfrom the detection array.

[0222] The software can be implemented by a processor 200 running on anetworked server or locally on a desktop computer. The processor isinterfaced with databases 210, 220, and 230. These databases can bestored in local memory, on machine-readable media, or on remote servers.The processor is also, directly or indirectly, interfaced with externalapparati, for example, an array synthesizer 240, an oligonucleotidesynthesizer 250, or a liquid handling robot 260.

[0223] The software can include a graphical user interface (GULI) thatdisplays known polymorphisms, e.g., SNPs for user selection. Thepolymorphisms can be pre-grouped based on relevance for variousdiagnostic, disease, or gene-mapping projects. The user can select oneor more of the groupings as desired.

[0224] Polymorphism information can be stored in a database 220 ofpolymorphisms. For each polymorphism, the database 220 can also indicateone or more precalculated items of information. Such information caninclude the availability one or more restriction enzymes which can beused to fragment genomic nucleic acid in order to produce a fragment ofdesired size. Multiple local restriction enzyme sites can be stored inorder to allow optimization of overall restriction enzyme selection suchthat enzymes that function in compatible buffers can be pooled. Thedatabase 220 can also store information about optimal SSPoligonucleotide target sites for each available restriction site.Alternatively, this information can be determined after polymorphismselection.

[0225] For example, the process 120 for identifying appropriaterestriction enzymes can include searching a database 210 of restrictionenzyme information to identify restriction enzymes that digest near apolymorphism site in order to produce a fragment of appropriate size.The restriction enzyme database 210 includes information about thespecific recognition sites of each enzyme, and its compatibility withvarious buffer conditions. The database can include, for example,information from the database that was established by Roberts et al.(Nucl. Acids. Res. 2001, 29:268-269) and includes information for over3000 enzymes.

[0226] After polymorphisms are selected, the system can output anoptimized combination of restriction enzymes to be used to fragment thesample nucleic acid. The software can, in some embodiments, also controla robotic system to prepare the determined restriction enzyme pool.

[0227] The system also designs one or more SSP oligonucleotides for eachpolymorphism. The system can optimize primer design for T_(m), e.g., soall target binding regions of a group of SSP oligonucleotides have asimilar T_(m), primer dimer formation, absence of palindromes, and soforth. The system can be interfaced with an oligonucleotide synthesizerto produce the SSP oligonucleotides or oligonucleotide array synthesizerto produce an array of immobilized SSP oligonucleotides.

[0228] Similarly, a related, or even the same system can be used toprocess information for nucleic acids detected on paired complementaryarrays. The system can be used to maintain a database that includes datarepresenting hybridization to a sense probe, and hybridization to anantisense probe, and relationships between the sense and anti-senseprobe. The database can also include a ratio between hybridizationlevels for a first and second target material to their correspondingsense probes and a ratio between hybridization levels for the first andsecond target material to their corresponding anti-sense probes. Asdescribed above, the hybridization material is appropriately generatedfor each probe set.

[0229] In another aspect, the invention features a system that providesaccess to a database that includes information about transcript levelsfor a plurality of genes. The database can include records that includea reference describing a sample (e.g., tissue source, tissue type and soforth), a reference to a profile (the profile being a table describingtranscript levels for the plurality of genes), and a locator indicatingthe identity or location of the support that includes archived templatesthat can be transcribed to produce aRNA or sRNA corresponding to thesample. The database can include at least ten records, e.g., eachreferring to a different mammalian tissue. In some embodiment, eachsample is microdissected. In some implementations, the insoluble supportcan be provided to a user (e.g., a customer) in combination with accessto the database, particularly to the record referring to that particularinsoluble support. Database access can be provided in a variety of ways,e.g., by distribution of an access code (e.g., for Internet access) orby distribution of a machine readable medium that includes the recordsthemselves.

[0230] Array Synthesis

[0231] Some embodiments use one or more arrays, for example: (1) anarray of SSP oligonucleotides; and (2) a detection array (e.g., apolymorphism or transcript detection array). An array can be aninsoluble support that includes a plurality of addresses. Each addresscan include a homogenous population of immobilized nucleic acids, e.g.,nucleic acids of predetermined sequence. The density of addresses can beat least 10, 50, 200, 500, 10³, 10⁴, 10⁵, or 10⁶ addresses per cm²,and/or no more than 10, 50, 100, 200, 500, 10³, 10⁴, 10⁵, or 10⁶addresses/cm. Addresses in addition to addresses of the plurality can bedeposited on the array. The addresses can be distributed, on thesubstrate in one dimension, e.g., a linear array; in two dimensions,e.g., a planar array; or in three dimensions, e.g., a three dimensionalarray. (e.g., layers of a gel matrix).

[0232] In one embodiment, the substrate is an insoluble or solidsubstrate. Potentially useful insoluble substrates include: massspectroscopy plates (e.g., for MALDI), glass (e.g., functionalizedglass, a glass slide, porous silicate glass, a single crystal silicon,quartz, UV-transparent quartz glass), plastics and polymers (e.g.,polystyrene, polypropylene, polyvinylidene difluoride,poly-tetrafluoroethylene, polycarbonate, PDMS, acrylic), metal coatedsubstrates (e.g., gold), silicon substrates, latex, membranes (e.g.,nitrocellulose, nylon). The insoluble substrate can also be pliable. Thesubstrate can be opaque, translucent, or transparent. In someembodiments, the array is merely fashioned from a multiwell plate, e.g.,a 96 or 384 well microtitre plate.

[0233] The array of SSP oligonucleotides has an SSP oligonucleotide ateach address such that the promoter is accessible and functional and thetarget binding region is able to specifically recognize the target site.In some embodiments, the 3′ terminus of the SSP oligonucleotide isextendable, e.g., by a DNA polymerase when hybridized to a template. TheSSP oligonucleotide can be anchored to the array substrate at the 5′terminus. Alternatively, the SSP oligonucleotide can be anchored to thearray substrate at a non-terminal nucleotide, so long as the abovepreconditions are satisfied. In other embodiments, the 3′ terminus isnon-extendable.

[0234] One method of anchoring SSP oligonucleotides requiressynthesizing an amino-modified nucleotide. During the phosphoramiditesynthesis, at the desired position, an amino-modified nucleotide isincluded. The resulting amino-modified SSP oligonucleotide is thendeposited on a surface activated to covalent couple to amino groups.Such a surface and method are described in provisional patentapplication, U.S. Serial No. 60/293,888, filed May 24, 2001. The surfaceis characterized by a covalently bonded activated group that includes anelectron-withdrawing group on an N-substituted sulfonamide.

[0235] A second method of anchoring SSP oligonucleotides requiressynthesizing the SSP oligonucleotides directly on an insoluble supportusing a 5′→3′ synthetic method, such as the method described in PCT US01/02689. This method provides nucleotide arrays having C-5′ bound tothe surface and C-3′ at the terminus. The arrays can be produced byreacting C-5′ activated, C-3′ photolabile group protected nucleotides,with a terminal hydroxyl group bound to the surface. After coupling amodified nucleotide to the surface, the C-3′ photolabile protectinggroup can be deprotected via a photochemical reaction to form a freehydroxyl group at the C-3′ terminus. The hydroxyl group, in turn, canreact with a modified nucleotide including a C-5′ phosphorous activatinggroup to tether the modified nucleotide to the surface. Repeatedselective coupling of modified nucleotides carrying a C-5′ phosphorousactivating group, such as phosphoramidite, and selectivephotodeprotection of the C-3′ photolabile protecting groups formsimmobilized oligonucleotides arrays having C-5′ attached to the solidsurface and the C-3′ at the terminal position. Selectivephoto-deprotection can be accomplished by several known methods, e.g.photolithography methods (as disclosed in Science (1991) 251:767-773;Proc. Natl. Acad. Sci. USA 93:13555-13560, (1996); U.S. Pat. Nos.5,424,186; 5,510,270; and 5,744,305, and 5,744,101) or a digitalmicromirror technique (e.g., as described in Sussman et. al. (1999)Nature Biotechnology 17:974-97).

[0236] A third method of forming an SSP array includes the deposition ofan unmodified oligonucleotide on a substrate. Numerous methods areavailable for dispensing small volumes of liquid onto substrates. Forexample, U.S. Pat. No. 6,112,605 describes a device for dispensing smallvolumes of liquid. U.S. Pat. No. 6,110,426 describes a capillaryaction-based method of dispensing known volumes of a sample onto anarray.

[0237] In addition to these exemplary methods, any of the applicablearray synthetic method can be used so long as the oligonucleotide isfunctional as a promoter and the target binding region is specific forthe target site.

[0238] The second type of array includes a plurality of detectionprobes. The probes can be designed in any of a number of formats todetect SNPs or mRNA. For example, a pair of probes can be used for eachbiallelic SNP. Each pair has the appropriate nucleotide at the queryposition to detect one of the two alleles. The query position can be atthe terminus of the detection probe. In another embodiment, thedetection probe is a primer, and base extension protocols, e.g., asdescribed in (Law and Brewer (1984) Proc. Natl. Acad. Sci. USA 81:66-70;Pastinen et al. (2000) Genome Res. 10: 1031-1042) are used to assesswhich allele is present. In still another embodiment, the query positionis more centrally located, and the detection probe can be used, forexample, as described in U.S. Pat. No. 5,968,740.

[0239] Uses

[0240] The methods and arrays described here can be used fortranscription amplification. One exemplary application is genotyping toinvestigate the presence of single nucleotide polymorphism (SNP) withina gene. The significance of SNPs is described in Weaver,“High-throughput SNP Discovery and Typing for Genome-wide GeneticAnalysis”, Trends in Genetics, December 2000). The detection ofpolymorphisms has a variety of applications. Non-limiting examplesinclude medical diagnostics, forensics, disease gene mapping,environmental management, agriculture, and protein evolution. Anotherexemplary application is evaluating transcripts in a sample, e.g., asample that includes cells, e.g., fewer than 10 000 or 1 000 cells.

[0241] mRNA Libraries

[0242] One exemplary application of the invention is the amplification,analysis and archiving of mRNA populations. This process enables thehigh throughput amplification and detection of mRNA from small amountsof starting material, e.g., less than 1 μg, 100 ng, 10 ng, or 1 ng. Forexample, the process can be used to profile the expression of genes in asingle cell. Further, the process results in an archive of the inputnucleic acid sample. The archive can be repeatedly transcribed, topermit analysis of the sample. The mRNA population can be amplifiedusing an immobilized oligonucleotide primer as a reverse transcriptionprimer, e.g., as described above.

[0243] The benefits of the application are numerous. For example, themethod does not require a number of manipulations such as precipitationsand spin column separations. The washing and exchange of solutions issimplified as the cDNA archive is immobilized on the insoluble support.Washing also removes unbound targets, such as ribosomal RNA, which caninterfere in reverse transcription by providing sites for non-specificpriming.

[0244] The insoluble support serves as a DNA archive of the originalmRNA sample. The archive can be returned to, time and again. Moreover,the archive is amplified by transcription, which restores the originalsample in its RNA state. Such amplification is also linear and may beless susceptible to biasing events than, e.g., exponentialamplification. In some embodiments, the method is supported by a singleprimer for reverse transcription. The primer is universal for allpolyadenylated mRNAs.

[0245] In one embodiment, the method is used to archive an mRNA samplefrom a limited number of cells, e.g., fewer than 100 cells, e.g., asingle cell. The DNA archive of the mRNA sample (or a sample of anynucleic acid) can be constructed for, e.g., normalized libraries,subtracted libraries and reduced complexity libraries.

[0246] RNA replicates generated from the insoluble support can be used,e.g., for profiling transcripts in the original mRNA sample, in vitrotranslating transcripts representative of transcripts in the originalmRNA sample, and generating dsRNA.

[0247] In one embodiment, RNA replicates (aRNA or sRNA replicates, asappropriate) are used in a subtractive hybridization reaction. Forexample, aRNA replicates from a first sample can be subtracted from sRNAreplicates produced from a second sample or from DNA produced from asecond sample. Methods for subtractive hybridization are well known(e.g., one set of replicates can be attached to an insoluble support).In one embodiment, the method includes two subtraction hybridizations,one forward (e.g., aRNA vs. sRNA), the other backward (e.g., sRNA vs.aRNA). The net result is a highly differential comparison.

[0248] All cited references, patents, and patent applications areincorporated by reference in their entirety. Accordingly, U.S.Application Serial No. 60/312,443, filed Aug. 15, 2001; 60/338,523,filed Nov. 5, 2001; 60/373,364, filed Apr. 16, 2002; Ser. No. 10/219,616filed Aug. 15, 2002; and Ser. No. 10/341,199, filed on Jan. 10, 2003,inclusive of any figures and sequence listings, are incorporated byreference in their entirety. The following examples illustrate thespecific embodiments of the invention described herein. As would beapparent to persons skilled in the arts, various changes andmodifications are possible and are contemplated within the scope of theinvention described.

EXAMPLE 1

[0249] Preparation of Aminopropylsilylated CPG Beads

[0250] 10 g CPG beads (906 A, 80-128 mesh) were heated at 80° C. for 3hr, cooled under nitrogen to the ambient temperature, and packed in a 25mm×120 mm glass wool insulated HPLC column. 80 mL of 1:73-aminopropyltriethoxysilane and dry toluene were heated to around from54° C. to 98° C. in a heated flask and then continuously pumped throughthe CPG beads packed column. The temperature of the column was monitoredat around 37° C. to 45° C. during the course of reaction of about 38 hr.After the reaction was completed, the packed CPG beads were washed inthe column twice with 125 mL methanol and twice with 125 mL acetone, andpoured into a glass container. After drying under high vacuum in theglass container, the beads were stored under nitrogen ready for nextsynthesis.

[0251] Preparation of 3-(3-(N-cyanoethyl-toluenesulfonamidocarbonyl)propyl-carboxamido-propylsilated CPG Beads

[0252] 0.5 g of the above aminopropylsilylated CPG beads, 200 mg of3-(toluenesulfonamido-carbonyl) propionic acid, 400 mg of Bop, and 4 mLdry NMP, and 0.25 mL diethylpropylamine were charged to a 6 mL reactionvial in a glove box. After shaking gently for about 3.5 hr, the CPGbeads were transferred to a filtering cartridge, drained, and washedtwice with 6 mL acetone, twice with 6 mL NMP, three times with 6 mLacetone. After drying under high vacuum, the beads were transferred to a6 mL reaction vial. The beads were then treated with 4 mL dry NMP and0.25 mL chloroacetonitrile and 0.25 mL diethylpropylamine, and shakengently at the ambient temperature for about 21 hr. The CPG beads weretransferred to a filtering cartridge, drained, and washed twice with 6mL NMP, four times with 6 mL methanol, and twice with 6 mL acetone.After drying under high vacuum for 2 hr, the CPG beads were ready forsolid phase covalent bonding reactions.

[0253] Experimental: CPG beads were activated. 100 pmols ofAmC6N12T7dT20V oligo primers were coupled onto 4 mg of CPG beads in 400mM sodium carbonate buffer at pH 9.5 for 1 hour at room temperature,followed by incubation overnight at 4° C. The beads were then blockedfor two hours in blocking buffer, which contain 50 mM ethanolamine, 0.1M Tris, and 0.1% SDS pH 9.0. After blocking, the beads were washedseveral times in Tris-buffered saline and either use immediately, orstore in 70% ethanol. cDNA synthesis and transcription were carried outas described.

[0254] The sequence of [AmC6]N12T7dT20V: is as follows:5′-[AmC6]ATAGGCGCGCCAATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTT (SEQ IDNO:28) TTTTTTTV-3′

[0255] Results: Covalently coupled promoter primers, which contain a PEGlinker and a 12 base spacer, were used successfully for templatepreparation and transcription from the CPG beads.

EXAMPLE 2 Covalent Coupling of TCR Primer to Microtitre Strips

[0256] TCR primer oligonucleotides with the following sequences weresynthesized. N12 Primer:5′-ATAGGCGCGCCAATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTTT (SEQ ID NO:29)TTTV-3′ N25 Primer:5′-ACGTACGTACGTCATAGGCGCGCCAATTAATACGACTCACTATAGGGAGATTTT (SEQ ID NO:30)TTTTTTTTTTTTTTTTV-3′ N50 Primer:5′-ACGTACGTACGTACGTACGTACGTCACGTACGTACGTCATAGGCGCGCCA (SEQ ID NO:31)ATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTTTTTTV-3′

[0257] Each oligonucleotide was phosphorylated at the 5′-end andpurified by HPLC method individually. Each 5′-phosphorylated primer wasthen diluted to 100 nM in 10 mM 1-methylimidazole (pH 7.0) and thesolution was added with 20 mM EDC. Each coating mixture was thenpipetted to NUCLEOLINK™ aminated strips (from Nalge Nunc International,Rochester, N.Y.): 100 μL per well. All the primer coupling reactionswere continued at 42° C. for 4 hours. Each empty well was washed once atroom temperature, soaked for 5 minutes at 42° C., and washed three moretimes at room temperature, all with 20 mM PBS (pH 7.4)/0.1% Tween20/0.5% bovine γ globulin (BgG.) To remove salt residues, the emptywells were washed three times with MILLIQ™ water (Millipore Corp,Billerica, Mass.). The dry primer coated strips were stored at 4° C.until use.

[0258] To investigate the binding activity of TCR primer coated on themicrotitre wells, samples of probe oligonucleotide of5′-Bt-GCGCCAATTATCGAAAAAAAAAAAAAAAAAA (SEQ ID NO:32) were prepared inPBS/0.1% Tween 20/0.5% BgG, at the following concentrations: 0, 0.28,0.56, 1.4, 2.8, 5.6, and 50 nM. The hybridization and detection assayswere carried out in triplicates according to the following procedure.

[0259] 1. Pipet 20 μL of biotinylated probe sample into each well.

[0260] 2. Incubate at 42° C. for 30 minutes.

[0261] 3. Wash 3 times with 100 μL of 2×SSC/0.1% Tween 20 buffer.

[0262] 4. Pipet 20 μL of Streptavidin-horse radish peroxidase conjugatesolution (Pierce Chemical), 0.2 μg/mL in PBS/0.05% Tween 20, into eachwell.

[0263] 5. Incubate at room temperature for 30 minutes.

[0264] 6. Wash once at room temperature, soak at 42° C. for 20 minutes,and then wash 3 more times, all with 100 μL of PBS/0.1% Tween 20/0.5%BgG.

[0265] 7. Pipet 50 μL of 1.5 mM o-phenylenediame in 1× stable substrate(Pierce Chemical), into each well.

[0266] 8. Incubate at room temperature for 15 minutes.

[0267] 9. Add 150 μL of 0.5M Sulfuric acid to each well.

[0268] 10. Measure absorbance at 492 nm on a microplate reader.

[0269] Results: The averaged absorbance from the triplicates of eachsample is shown in the Table 2 below. TABLE 2 Detection of ImmobilizedOligonucleotides Probe Conc. (nM) Absorbance 0 0.015 0.28 0.835 0.561.303 1.4 1.708 2.8 2.094 5.6 2.224

[0270] This dose dependency of biotin probe concentration demonstrated arobust assay with 3 log-orders of dynamic range and also specific targetbinding activity for the primer immobilized on the insoluble support.

[0271] The microtitre strips coated with three different TCR primeroligonucleotides were used to amplify human liver RNA according to thefollowing procedure.

[0272] 1. 500 ng human liver RNA (Ambion, Inc. Austin, Tex.) wasannealed to insoluble support anchored oligos in the presence of firststrand synthesis buffer and DNase inhibitor was used in each of the twopositive controls (1 μg per reaction.) The negative control was a wellto which no RNA was added. mRNAs were annealed at 42° C. for 5 minutes.

[0273] 2. cDNA synthesis was initiated by adding sodium pyrophosphate,and AMV reverse transcriptase. (Promega Catalog No. C4360)

[0274] 3. Reactions were incubated at 42° C. for 1 hour.

[0275] 4. Second strand cDNA synthesis was initiated by the addition of40 μL of 2.5×second strand synthesis buffer (1×=40 mM Tris-HCl, pH 7.2);5 μL of 1 mg/mL acetylated BSA; 23 units of DNA polymerase 1; 0.8 unitof RNase H, and nuclease free water to final volume of 100 μL. Incubatedat 14-16° C. for 2 hours.

[0276] 5. Wells were washed several times with 50 mM Tris-HCl, pH 8.0.The wells were then placed on ice, and 20 μL 1×T7 RNA polymerasetranscription buffer was added.

[0277] 6. Transcription reactions were performed in 20 μL volume, byfollowing protocol provided by the manufacturer (AMPLISCRIBE™ T7 HIGHYIELD TRANSCRIPTION KIT, Cat No. AS2607, Epicentre, Madison, Wis.)Reactions were incubated at 37° C. for 1-2 hours.

[0278] 7. 5 μL of the reactions was analyzed on an agarose gel.

[0279] Results: Agarose gel analysis revealed that RNA with a mediumdistribution between 0.4-1.0 kb was amplified by the N25 and N50 primerscoated wells. N12 primer coated wells gave some amplification of RNA,but less than the amount by the N25 and N50 primers. See Table 3 below.No RNA transcripts were amplified by the negative controls. TABLE 3Purified RNA Estimated Amplification N12 Primer Coated Wells 0.35 μg  35fold N25 Primer Coated Wells 1.91 μg 191 fold N50 Primer Coated Wells1.51 μg 151 fold

[0280] It was determined that the N25 primer coated wells repeatedlygave better amplification of RNA than N50 and N12 primers. The optimallength of the spacer is 25 nucleotides or a linker of equivalent lengthformed by a chemical substitute.

Example 3 Biotinylated Primer Length

[0281] In these experiments, 5′ biotinylated oligonucleotide primerscontaining a 0, 6, or 12 nucleotides spacer sequence between the biotinand the promoter sequence, were tested for their effects ontranscription. Reaction conditions for cDNA and for transcription weresimilar to previously described. Oligo-primer densities tested were 10pmols per well and 1 pmol per well. The amount of RNA used was,variously, 1 microgram and 500 ng per reaction.

[0282] Enhanced amplification was observed with the primers that include6 and 12 nucleotide spacers (Bt(6)T7dT20V and Bt(12)T7dT20V) relative tothe amplification observed with the primer having a 0 nucleotide spacer(Bt(0)T7dT20V).

[0283] Primer Sequences: Bt(0)T7dT20V:5′-Bt-ATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTTTTTTV 3′ (SEQ ID NO:33)Bt(6)T7dT20V: 5′-Bt-GCGCCAATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTTTTTTV3′ SEQ ID NO:34 Bt(12)T7dT20(V):5′-Bt-ATAGGCGCGCCAATTAATACGACTCACTATAGGGAGATTTTTTTTTTTTTTTT (SEQ IDNO:35) TTTTV-3′

EXAMPLE 4

[0284] The solution mode of sequence specific amplification bytranscription was used to amplify the Stul fragment of the humanapolipoprotein E gene. (This fragment codes for amino acid 72 to aminoacid 209).

[0285] Human genomic DNA was purchased from Sigma Chemicals (St. Louis,Mo.). Samples of 20 μl containing 10 μg high molecular weight human DNAwere digested for 3 hours at 37° C., using 10 units of StuI (New EnglandBiolabs, Beverly, Mass.). The StuI digests were diluted to 50 μl in thepresence of 1× lambda exonuclease buffer (67 mM Glycine-KOH(pH 9.4), 2.5mM MgCl₂, and 50 μg/ml BSA), and 10 units of lambda exonuclease (NewEngland Biolabs, Beverly, Mass.) and incubated at 37° C. for 30 minutes.This enzyme reaction was terminated by incubation at 75° C. for 30minutes.

[0286] Subsequently, the reaction mixture was further diluted to 100 μlin the present of 1×Klenow fragment (3′->5′ exo⁻) buffer (10 mM Tris-HCl(pH 7.5), 5 mM MgCl₂, 7.5 mM dithiothreitol), and 10 pmols of SSPoligonucleotide (SEQ ID NO: 1; T7StuSE), and hybridization was carriedout at 37° C. for 10 minutes. The SSP oligonucleotide anneals to one endof the StuI fragment of the human apolipoprotein E gene (amino acid 72to amino acid 209). The following is the sequence of the T7StuSE:5′-AATTAATACG ACTCACTATA GGGAAGGCCT ACAAATCGGA ACTGGAG-3′ (SEQ ID NO:1)

[0287] The T7 polymerase promoter is underscored. The apoE annealingsite is 3′ to the promoter.

[0288] After SSP oligonucleotide annealing, the primer and apoE targetare extended by the addition of 10 units of Klenow fragment DNApolymerase (New England Biolabs, Beverly, Mass.), 10 mM each of dATP,dGTP, dTTP, and dCTP during incubation at 37° C. for 1 hour. After heatinactivation of the enzyme at 75° C. for 30 minutes, the mixture wasadjusted to 2.5 M ammonium acetate, and two volumes of 100% ethanol wereadded to precipitate the DNA. DNA was then recovered by centrifugationand dissolved in 20 μl of 10 mM Tris-HCl at pH 8.0.

[0289] The apoE target was then amplified by transcription. An aliquotof the ethanol precipitated DNA was in vitro transcribed using theAMPLISCRIBE™ T7 transcription kit from Epicentre (Madison Wis.). Theresulting transcription products were analyzed by agarose gelelectrophoresis. RNA products of the expected size were observed only inSSP oligonucleotide extended genomic DNA, and were absent in controlsfrom unprimed genomic DNA.

[0290] Gel electrophoresis of the following samples validated themethod. Lane 1: 100 bp DNA marker; Lane 2: 10% of the T7 transcriptionreaction from 250 ng of lambda exonuclease treated, human genomic DNA;Lane 3: 10% of the T7 transcription reaction from 250 ng of lambdaexonuclease treated, SSPP primer extended human genomic DNA; Lane 4: 10%of the T3 transcription reaction from same DNA as in Lane 3; Lane 5: 10%of the T7 transcription reaction from 60 ng of clone apoE DNA andtreated as in Lane3; Lane 6: 10% of the T3 transcription reaction fromsame DNA as in Lane 5; Lane 7: 10% of the T7 transcription reaction fromapoE clone, no treatment; Lane 8: 1 μg human genomic DNA, no treatment

[0291] To confirm that in-vitro transcribed RNA is indeed apoE RNA,RT-PCR was performed according to the protocol provided by the vendor(THERMOSCRIPT™ RT PCR systems, Life Technologies, Bethesda, Md.). PCRusing the primer pair, P3 and P6ASE (SEQ ID NO:2 and SEQ ID NO: 3),produced the correct size product, only for DNA derived from RNAtranscribed from SSP oligonucleotide-extended genomic DNA. There is noPCR product derived from the RNA transcription mixture, suggesting thePCR product is not from unprimed genomic DNA. There is no PCR productwhen using the primer pair T7 and P6ASE (SEQ ID NO:4 and SEQ ID NO:3)confirming the PCR template is indeed cDNA derived from RNA.

[0292] Gel electrophoresis of controls and test reactions validatedmethod. A specific amplified fragment was evident when human genomic DNAwas used as the template with the appropriate primers. The amplified RNAwas detected by PCR. Further, he PCR product was isolated frompreparative agarose gel electrophoresis, and sequenced. DNA sequencingconfirmed that the PCR product was indeed apoE.

[0293] These reactions and manipulations can be coupled and streamlinedto achieve considerable gains in efficiency and economy.

EXAMPLE 5

[0294] SSAT is suited for multiplex reactions. In this example, multipletarget fragments were amplified by site specific amplification bytranscription. A 5.5 kb genomic human apoE serve as DNA target templatein this manipulation. Briefly, apoE DNA is cleaved by restrictionenzymes AvaI, BsrD1, and Stul (New England Biolabs, Beverly, Mass.) togenerate eight DNA fragments. One of the eight DNA fragment is the sameStul fragment as described previously in Example 1. The human apoEsequence is available, e.g., from GenBank entry AF 261279.

[0295] The primer sequences listed in SEQ ID NOS:1 to 22 were used: SEQID NO:1 (T7StuSE): AATTAATACG ACTCACTATA GGGAAGGCCT ACAAATCGGA ACTGGAGSEQ ID NO:2 (P3): GAACAACTGA CCCCGGTGGC GG SEQ ID NO:3 (P6ASE):GAGGCGAGGC GCACCCGCAG SEQ ID NO:4 (T7): TTAATACGAC TCACTATAGG G SEQ IDNO:5 (T7AvaSE2): CATTAATACGACTCACTATAGGGACTCGGGGTCGGGCTTGGGGAGA SEQ IDNO:6 (T7AvaSE3): CATTAATACGACTCACTATAGGGACCCGGGAGAGGAAGATGGAATTTTC SEQID NO:7 (T7AvaSE4): CATTAATACGACTCACTATAGGGACCCGAGCTGCGCCAGCAGACCGAG SEQID NO:8 (T7BsrD1SE): CATTAATACGACTCACTATAGGGACATTGCAGGCAGATAGTGAATACCSEQ ID NO:9 (T7stuSE2): CATTAATACGACTCACTATAGGGAAGGCCTGGGGCGAGCGGCT SEQID NO:10 (T7StuSE3): CATTAATACGACTCACTATAGGGAAGGCCTTCCAGGCCCGCCTCAAGASEQ ID NO:11 (AvaSE2): CTCGGGGTCGGGCTTGGGGAGA SEQ ID NO:12 (AvaSE3):CCCGGGAGAGGAAGATGGAATTTTC SEQ ID NO:13 (AvaSE4):CCCGAGCTGCGCCAGCAGACCGAG SEQ ID NO:14 (BsrD1SE):CATTGCAGGCAGATAGTGAATACC SEQ ID NO:15 (StuSE2): AGGCCTGGGGCGAGCGGCT SEQID NO:16 (StuSE3): CCTTCCAGGCCCGCCTCAAGA SEQ ID NO:17 (AvaASE2):CCCAGTAGGTGCTCGATAAATG SEQ ID NO:18 (AvaASE3): AGAAGAGGGGGCCCAGGGTCTGSEQ ID NO:19 (AvaASE4): TGAGTCAGAAGGGAAGAGAGAGAG SEQ ID NO:20(BsrD1ASE): AGCACAGGTGTGTGGCACCATG SEQ ID NO:21 (StuASE2):CTCGTCCAGGCGGTCGCGGGT SEQ ID NO:22 (StuASE3): TCCACCCCAGGAGGACGGCTG

[0296] 10 μg of a plasmid DNA containing the 5.5 kb human apoE gene wasdigested with 40 units of Ava1 and 40 units of Stu1 for 4 hours at 37°C. Subsequently, 20 units of BsrD1 was added to the reaction mixture.Incubation was continued for an additional 2 hours at 65° C. Therestriction digestion was quenched on ice. apoE DNA fragments werepurified by the mini-elute enzyme clean-up kit (QIAGEN Inc.). An aliquotof 2 μg of the restricted DNA was treated with 2 units of lambdaexonuclease at 37° C. for 30 minutes. The exonuclease reaction wasterminated and inactivated by incubation at 80° C. for 15 minutes. Thereaction mixture was adjusted to contain 2.5 M ammonium acetate, andprecipitated by the addition of 2.5 volumes of 100% ethanol. Theresulting mixture was then incubated on ice for two hours, and thencentrifuge at room temperature at 16,000×g for 15 minutes in a BeckmanALLEGRA™ micro-centrifuge to pellet the DNA. The ethanol supernatant wasremoved by pipetting, and the DNA pellet was rinsed with 70% ethanol,air dried, and dissolve in sterile MILLIQ™ water (Millipore Corp,Billerica, Mass.).

[0297] Primer annealing was carried out in 30 μl containing 1×Klenow(3′-5′exo⁻) buffer, 50 pmols of each T7apoE sequence primers (SEQ ID NO:1, 5, 6, 7, 8, 9 and 10) and 1.8 μg of the lambda exonuclease treatedDNA. The reaction mixture was heated at 75° C. for 5 minutes, followedby incubation at 37° C. for another 10 minutes. The annealing mixturewas then diluted to 50 μl 1 with 1×Klenow buffer in the presence of 1 mMdNTPs and 10 units of Klenow enzyme (3′-5′ exo⁻) for 1 hour at 37° C.The extension reaction was terminated by heating at 75° C. for 20minutes to inactivate the enzyme. Excess T7apoE sequence primers wereremoved by Exonulease1 (Amersham Pharmacia Inc.). Exonuclease1 wasremoved by the mini-elute enzyme purification kit as described earlier.

[0298] To detect the SSP oligonucleotide-extended product, an aliquot ofthe treated DNA was in vitro transcribed using the AMPLISCRIBE™ T7transcription kit from Epicentre, (Madison, Wis.). The RNA was thenreverse transcribed into cDNA using antisense apoE primers specific foreach of the seven restriction fragments (SEQ ID NO:3, 17, 18, 19, 20, 21& 22, respectively). Only the RNA which includes the corresponding sensestrand of the apoE reverse transcription primer sequences were reversetranscribed into cDNA. cDNA synthesis reaction was carried out accordingto the protocol of the THERMOSCRIPT™ RT PCR system (Life Technologies,Bethesda, Md.). PCR reactions were carried out in 20 μl volume, in anEppendoff DNA thermocycler, using AMPLITAG™.

[0299] Gold DNA polymerase and apoE sequence primer pairs which arespecific for the seven target restriction fragments (SEQ ID NO:2,3,11-22.). A PCR assay detected amplified products from all RNA-amplifiedapoE fragments: AvaI-2, AvaI-3, AvaI-4, BsrD1, StuI-1, StuI-2, andStuI-3. All seven primer pairs amplified a prominent band of theexpected size from cDNA but not from RNA. DNA sequencing of fiverepresentative fragments confirmed that they are all correct apoEsequences.

EXAMPLE 6 Solid Phase Based Amplification

[0300] This example illustrates solid-phase sequence specificamplification by transcription. The Stul fragment of humanapolipoprotein E gene (encoding amino acid 72 to amino acid 209) is thetest substrate model. The following primer sequences were used: SEQ IDNO: 2, SEQ ID NO: 3, and SEQ ID NO: 23.

[0301] Streptavidin coated microplates (NoAb Biodiscoveries,Mississauga, Ontario, Canada) were used to immobilize 5′ end biotinlabeled, SSSP oligonucleotide oligonucleotide (BT7StuSE1). Briefly,oligos were diluted to 100 μl with TBS (Tris-Buffered Saline, 20 mMTris, 500 mM sodium chloride, pH 7.5), and spotted into 8-well strips.Each well contained either 50 pmols or 5 pmols of BT7StuSE1. Thenegative-control well contained 50 pmols of T7StuSE1 oligo (SEQ IDNO: 1) primer. After a two hour incubation at room temperature,oligonucleotide solutions were discarded, and the wells were rinsedseveral times with 100 μl TBS, 0.01% Tween 20, followed by incubation atroom temperature for 30 minutes with a blocking solution of TBS, 0.01%Tween 20 and 100 μg/ml BSA (New England Biolabs, Beverly, Mass.). Thewells were then rinse twice, with 100 μl TBS followed by 100 μl ofKlenow buffer (3′-5′ exo⁻). Single-stranded DNA were created by lambdaexonuclease digestion of the restricted human Apo E fragments, aspreviously described in examples 1 and 2. 1 μg of the single-strandedDNA was annealed to the immobilized primers for 15 minutes at 37° C. in30 μl of Klenow buffer, followed by primer-extension in 100 μl reactionvolume that contained Klenow buffer, 1 mM dNTPs, and 20 units of KlenowDNA polymerase (3′-5′ exo⁻). Enzyme incubation was for one hour at 37°C. in a humidity chamber. The wells were then washed several times with200 μl of sterile 10 mM Tris-HCl, pH 8.0, and once, with 50 μl 1×T7 RNApolymerase transcription buffer. In-vitro transcription was carried outin 20 μl at 37° C. for two hours using the T7 AMPLISCRIBE™ kit fromEpicentre Technologies (Madison, Wis.). The resulting transcriptionproducts were analyzed by agarose gel electrophoresis. RNA product ofthe expected size was observed only in wells, which containedimmobilized Bt7StuSE1 primer, and is absent from the well whichcontained the T7Stu1 oligonucleotide primer. Furthermore, repeatable,robust transcription was maintained over a period of several days ofstorage at 4° C. After an initial round of transcription, the insolublesupport was stored at least for 24 hours, and then removed for furthertranscription. Five such reactions over a storage period of ten daysprovided continued amplification with no loss in yield.

[0302] To confirm the in-vitro transcribed RNA is indeed apoE RNA,RT-PCR was performed according to the protocol provided by THERMOSCRIPT™RT PCR systems (Life Technologies, Bethesda, Md.). To confirm the PCRproduct is indeed apoE sequence, the PCR product was isolated frompreparative agarose gel electrophoresis and sequenced. DNA sequencingconfirmed the PCR product was indeed apoE.

EXAMPLE 7

[0303] Human whole genomic DNA was treated with lambda exonucleases,hybridized to SSP primers attached to an insoluble support, extendedusing a DNA polymerase, then amplified in accordance with the singlepromoter SP-TCR method. Robust transcription was observed using inputhuman genomic DNA in an amount between 100 ng and 2 μg. The detection ofRNA amplified transcripts from the long sample is indicative of theunexpected amplification yield provided by the method. No RNA amplifiedproducts were detected in a negative control reaction. The reactionproduct migrated as a discrete band on agarose gel.

EXAMPLE 8

[0304] Materials for this example included:

[0305] mRNA: Human liver Poly A RNA purchased from Ambion Inc., Austin,Tex.

[0306] Anchor primer: Bt-T7d(T)₁₇V where Bt=5′biotin; T7=T7 RNApolymerase promoter; d (T) 17=a homopolymer of 17 T residue; V=A, G, andC. This primer has the sequence:5′-TTAATACGACTCACTATAGGGTTTTTTTTTTTTTTTTTV-3′ (SEQ ID NO:26)

[0307] Solid phase: streptavidin coated wells (NoAb Biodiscoveries,Mississauga, Ontario, Canada)

[0308] The procedure was as follows:

[0309] 1. 200 pmol Anchor Primer was attached each streptavidin coatedwell,

[0310] 2. Wells were washed with TBS and rinsed with 1×first strandsynthesis buffer,

[0311] 3. 2 μg human liver mRNA (Ambion, Inc. Austin, Tex.) was annealedto anchored oligos in the presence of first strand synthesis buffer, andDNase inhibitor (Universal Riboclone cDNA Synthesis System Catalog No.C4360, Promega Corp, Madison, Wis., USA). Kanamycin mRNA (Promega Corp)was used as a positive control (1 μg per reaction). The negative controlwas a well to which no RNA was added. mRNAs were annealed at 42° C. for5 minutes.

[0312] 4. cDNA synthesis was initiated by adding sodium pyrophosphate,and AMV reverse transcriptase (All reagents from Promega Catalog No.C4360). The final concentrations for all the components were: 1×firststrand synthesis buffer (50 mM Tris-HCL, pH 8.3 at 42 degree C.; 50 mMKCL; 10 mM MgCl2; 0.5 mM spermidine; 10 mM DTT; 1 mM each dATP, dCTP,dGTP, dTTP); 40 units of Rnasin ribonuclease inhibitor; 4 mM sodiumpyrophosphate and 30 units of AMV reverse transcriptase. The finalvolume of first strand cDNA synthesis reaction was 20 μl.

[0313] 5. Reactions were incubated at 42° C. for 1 hour.

[0314] 6. Second strand cDNA synthesis was initiated by the addition of40 μl of 2.5× second strand synthesis buffer (1×=40 mM Tris-HCL, pH7.2); 5 μl of 1 mg/ml acetylated BSA; 23 units of DNA polymerase 1; 0.8unit of RNase H, and nuclease free water to final volume of 100 μl.Incubate at 14-16° C. for 2 hours.

[0315] 7. 2 units of T4 DNA polymerase/μg of input RNA was added.Incubation was continued at 37° C. for 10 minutes.

[0316] 8. Wells were washed several times with 50 mM Tris-HCL, pH 8.0.The wells were placed on ice, and 20 μl 1× T7 RNA polymerasetranscription buffer was added.

[0317] 9. Transcription reactions were performed in 20 μl volume, byfollowing protocol provided by the manufacturer (AMPLISCRIBE™ T7 highyield transcription kit, Cat# AS2607, Epicentre, Madison, Wis.).Reactions were incubated at 37° C. for 1-2 hours.

[0318] 10. 5 μl of the reactions was analyzed on an agarose gel.

[0319] Results: A distribution of nucleic acid fragments correspondingto RNA transcripts of >0.4 kb, with a medium distribution between0.4-1.0 kb were observed in both reaction primed by human liver mRNAprimed cDNA library, and the reaction primed by kanamycin resistancegene mRNA. No RNA transcripts were detected from the negative control.

[0320] Nucleic acid was amplified by RT-PCR from the transcriptionreaction product produced from the insoluble substrate. Specific sizefragments corresponding to mRNAs for human serum albumin, beta-actin,and G3PDH were detected in the sample derived from the human liver mRNAsample, but not from the control sample of mRNA for the kanamycinresistance gene. Similarly, a nucleic acid fragment for the kanamycinresistance gene was detected in this control, whereas the liver specifictranscripts were not. This example demonstrated that mRNA can beamplified from an insoluble support prepared as described.

EXAMPLE 9

[0321] Materials for this example included:

[0322] mRNA: Human liver total RNA, and yeast RNA (Ambion Inc., AustinTex.). Kanamycin resistance gene control mRNA (Promega Corp).

[0323] Anchor primers: 1) Bt-T7d(T)₁₇V (see above). 2) Bt-ASC 1T3 whereBt=5′ biotin; T3=T3 promoter sequence; ASC1=restriction endonucleaserecognition site for AscI (GGCGCGCC). 3) TCR-adapter

[0324] Solid phase: streptavidin coated wells (NoAb Biodiscoveries,Mississauga, Ontario, Canada)

[0325] The first part of the procedure was as follows:

[0326] 1. 200 pmol Anchor Primer was attached each streptavidin coatedwell,

[0327] 2. Wells were washed with TBS and rinse with 1× first strandsynthesis buffer,

[0328] 3. Samples were annealed to anchored oligos in the presence offirst strand synthesis buffer, and DNase inhibitor (Universal RiboclonecDNA Synthesis System Catalog No. C4360, Promega Corp, Madison Wis.).Four separate reactions were set up. The reaction samples were: (a) 20μg human liver total RNA; (b) 20 μg human liver total RNA+1 ng kanamycinmRNA; (c) 20 μg human liver total RNA+10 ng kanamycin mRNA; and (d) 20μg yeast RNA+100 ng kanamycin mRNA K. mRNAs were annealed at 42° C. for5 minutes.

[0329] 4. cDNA synthesis was initiated by adding sodium pyrophosphate,and AMV reverse transcriptase (All reagents from Promega Catalog No.C4360). The final concentrations for all the components were: 1× firststrand synthesis buffer (50 mM Tris-HCL, pH 8.3 at 42 degree C.; 50 mMKCL; 10 mM MgCl2; 0.5 mM spermidine; 10 mM DTT; 1 mM each dATP, dCTP,dGTP, dTTP); 40 units of Rnasin ribonuclease inhibitor; 4 mM sodiumpyrophosphate and 30 units of AMV reverse transcriptase. The finalvolume of first strand cDNA synthesis reaction was 20 μl.

[0330] 5. Reactions were incubated at 42° C. for 1 hour.

[0331] 6. Second strand cDNA synthesis was initiated by the addition of40 μl of 2.5× second strand synthesis buffer (1×=40 mM Tris-HCL, pH7.2); 5 ul of 1 mg/ml acetylated BSA; 23 units of DNA polymerase 1; 0.8unit of DNase H, and nuclease free water to final volume of 100 μl.Incubate at 14-16 degree C. for 2 hours.

[0332] 7. 2 units of T4 DNA polymerase/ug of input RNA were added.Incubation was continued at 37° C. for 10 minutes.

[0333] 8. Wells were washed several times with 50 mM Tris-HCL, pH 8.0.9. TCR-Adapter ligation: Adapter ligation was performed in 30 ul volumeusing the Fast-link DNA ligation kit (Epicentre Cat#LK0750H, MadisonWis.). The final concentration of all the components were: 1× ligationbuffer (33 mM Tris-acetate pH 7.8, 66 mM potassium acetate, 10 mMmagnesium acetate, 5 mM DTT); 05 mM ATP; 20 pmol TCR-adapter. Incubateat room temperature for 30 minutes, and wash well several times with 50mM Tris-HCL pH 8.0 followed by 20 μl of 1×T7 transcription buffer.

[0334] 10. Transcription reactions were performed in 20 μl volumefollowing the protocol provided by the manufacturer (AMPLISCRIBE™ T7high yield transcription kit, Cat# AS2607, Epicentre, Madison, Wis.).Reactions were incubated at 37° C. for 1-2 hours.

[0335] 11. 4 μl of each reaction were analyzed on an agarose gel.

[0336] Result: The following RNA amplification products were observed:RNA transcripts >0.4 kb in length, with a medium distribution between0.4-1.0 kb were observed in all reactions that amplified human livertotal RNA spiked with 0, 1, or 10 ng of kanamycin mRNA), and a discreteRNA band was observed in reaction #4 which is derived from the controlmRNA for the kanamycin resistance gene.

[0337] These findings indicate that a cDNA library can be synthesized onan insoluble substrate from 20 μg of input human total RNA (whichcorresponds to approximately 200-400 ng of mRNA). The library can beamplified by transcription.

[0338] An individual species of 1 ng or less can be detected by thismethod as demonstrated by detection of the spiked RNA for the kanamycinresistance gene. Further, the library was stored for several days underrefrigeration. The stored library was effectively transcribed, thusverifying the value of this technique for archiving RNA populations. Itwas also found that the stored library could be effectively transcribedafter two or more months of storage.

EXAMPLE 10

[0339] RNA (16 μl) from the first T7 transcription (Example 6) wasprecipitated with ethanol and redissolved in 20 μl of nuclease-freewater. 4 μl of RNA were used for T3 transcription cycling (TCR). Theexperimental procedures were as follows:

[0340] 1. 200 pmol of the Bt-T3ASCl oligonucleotide was attached tostreptavidin coated wells

[0341] 2. Wells were washed with TBS and rinsed with 1×first strandsynthesis buffer

[0342] 3. The mRNA was annealed to the anchored oligonucleotides in thepresence of first strand synthesis buffer, and DNase inhibitor. A totalof 4 reactions were set-up as follows:

[0343] a) 4 μg RNA from T7 reaction 1 of Example 6;

[0344] b) 4 μg RNA from T7 reaction 2 of Example 6

[0345] c) 4 μg RNA from T7 reaction 3 of Example 6

[0346] d) 4 μg RNA from T7 reaction 4 of Example 6

[0347] Annealing was performed at 42° C. for 5 minutes.

[0348] 4. cDNA synthesis was initiated by adding sodium pyrophosphate,and AMV reverse transcriptase. The final concentrations for all thecomponents was: 1× first strand synthesis buffer (50 mM Tris-HCL, pH 8.3at 42° C.; 50 mM KCL; 10 mM MgCl₂; 0.5 mM spermidine; 10 mM DTT; 1 mMeach DATP, dCTP, dGTP, dTTP); 40 units of Rnasin ribonuclease inhibitor;4 mM sodium pyrophosphate and 30 units of AMV reverse transcriptase. Thefinal volume of the first strand cDNA synthesis reaction was 20μl.

[0349] 5. The reaction was incubated at 42° C. for 1 hour.

[0350] 6. Second strand cDNA synthesis was effected by the addition of40 μl of 2.5× second strand synthesis buffer (1×=40 mM Tris-HCL, pH7.2); 5 ul of 1 mg/ml acetylated BSA; 23 units of DNA polymerase 1; 0.8unit of DNase H, and nuclease free water to final volume of 100 μl.Incubate at 14-16° C. for 2 hours.

[0351] 7. Then 2 μl (6 units) of E. coli ligase was added to each well,and the incubation was extended for 30 minutes at 16° C.

[0352] 8. 2 units of T4 DNA polymerase/μg of input RNA was added to eachwell, and the incubation was extended for 10 minutes at 37° C.

[0353] 9. After the incubation, wells were washed several times with 50mM Tris-HCL, pH 8.0.

[0354] 10. Then transcription was used to amplify RNA from each well.Transcription reactions were performed in 20 μl following the protocolof AMPLISCRIBE™ T3 High Yield Transcription Kit, (Cat# AS2603,Epicentre, Madison Wis.). Reactions were incubated at 37° C. for 1-2hours.

[0355] 11. The reaction was analyzed by agarose gel electrophoresis.

[0356] Results: Transcription products having a size of 0.4-1 kb wereobserved in all 4 reactions. These results demonstrated the TCR adapterligated to the ends of the T7d(T)₁₇V primed cDNA from Example 9 waseffective for driving T3 DNA polymerase mediated amplification. Thisexample is a successful application of the so-called Transcription ChainReaction (TCR) method.

[0357] The incorporation of a rare sequence cutter restriction enzymesuch as AscI (which cuts human DNA on average once per 670,000 basepairs) permitted the release of the anchored library cDNA from theinsoluble support, thereby providing flexibility in downstreamapplications. It was also found that three full cycles of TCRamplification had an amplification power of greater than 10⁸, and that 1ng of total RNA was successfully amplified.

EXAMPLE 11

[0358] An exemplary dG tailing procedure is as following:

[0359] 1. Immobilize Btl 2T7d(T)20V anchor primer

[0360] 2. Anneal total RNA

[0361] 3. First strand synthesis as described

[0362] 4. Rinse three times with 50 mM Tris-HCL (pH 8.0), followed byrinsing with terminal transferase buffer.

[0363] 5. Add terminal transferase buffer, 25 μM dGTP, 10 units ofterminal transferase in 20 μl reaction, and incubate at 37° C. for 15minutes

[0364] 6. Wash wells and proceed to Tag annealing

[0365] Tag annealing and second strand synthesis:

[0366] 1. Combine 20 pmol Tag (LITP-1) in 20 μl EcoPoll buffer (NewEngland Biolabs, Beverly, Mass.), and incubate at 37° C. for 10 minutes

[0367] 2. Add dNTPs to final 1 mM; 5 ug nuclease free BSA; 0.8 units ofRnaseH, and 1 unit of Klenow enzyme in final volume of 50 μl. Incubatefor 1 hour at 37° C.

[0368] Transcription of the immobilized template produced by this methodis as described.

[0369] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1 35 1 47 DNA Artificial Sequence Synthetically generatedoligonucleotide 1 aattaatacg actcactata gggaaggcct acaaatcgga actggag 472 22 DNA Artificial Sequence Synthetically generated oligonucleotide 2gaacaactga ccccggtggc gg 22 3 20 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 3 gaggcgaggc gcacccgcag 20 4 21 DNA ArtificialSequence Synthetically generated oligonucleotide 4 ttaatacgac tcactataggg 21 5 46 DNA Artificial Sequence Synthetically generatedoligonucleotide 5 cattaatacg actcactata gggactcggg gtcgggcttg gggaga 466 49 DNA Artificial Sequence Synthetically generated oligonucleotide 6cattaatacg actcactata gggacccggg agaggaagat ggaattttc 49 7 48 DNAArtificial Sequence Synthetically generated oligonucleotide 7 cattaatacgactcactata gggacccgag ctgcgccagc agaccgag 48 8 48 DNA ArtificialSequence Synthetically generated oligonucleotide 8 cattaatacg actcactatagggacattgc aggcagatag tgaatacc 48 9 43 DNA Artificial SequenceSynthetically generated oligonucleotide 9 cattaatacg actcactatagggaaggcct ggggcgagcg gct 43 10 48 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 10 cattaatacg actcactata gggaaggcct tccaggcccgcctcaaga 48 11 22 DNA Artificial Sequence Synthetically generatedoligonucleotide 11 ctcggggtcg ggcttgggga ga 22 12 25 DNA ArtificialSequence Synthetically generated oligonucleotide 12 cccgggagaggaagatggaa ttttc 25 13 24 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 13 cccgagctgc gccagcagac cgag 24 14 24 DNAArtificial Sequence Synthetically generated oligonucleotide 14cattgcaggc agatagtgaa tacc 24 15 19 DNA Artificial SequenceSynthetically generated oligonucleotide 15 aggcctgggg cgagcggct 19 16 21DNA Artificial Sequence Synthetically generated oligonucleotide 16ccttccaggc ccgcctcaag a 21 17 22 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 17 cccagtaggt gctcgataaa tg 22 18 22 DNAArtificial Sequence Synthetically generated oligonucleotide 18agaagagggg gcccagggtc tg 22 19 24 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 19 tgagtcagaa gggaagagag agag 24 20 22 DNAArtificial Sequence Synthetically generated oligonucleotide 20agcacaggtg tgtggcacca tg 22 21 21 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 21 ctcgtccagg cggtcgcggg t 21 22 21 DNAArtificial Sequence Synthetically generated oligonucleotide 22tccaccccag gaggacggct g 21 23 19 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 23 taatacgact cactatagg 19 24 19 DNAArtificial Sequence Synthetically generated oligonucleotide 24aattaaccct cactaaagg 19 25 19 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 25 atttaggtga cactataga 19 26 39 DNAArtificial Sequence Synthetically generated oligonucleotide 26ttaatacgac tcactatagg gttttttttt ttttttttv 39 27 33 DNA ArtificialSequence Synthetically generated oligonucleotide 27 gcgccaattatcgaaaaaaa aaaaaaaaaa aaa 33 28 58 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 28 ataggcgcgc caattaatac gactcactat agggagattttttttttttt tttttttv 58 29 58 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 29 ataggcgcgc caattaatac gactcactat agggagattttttttttttt tttttttv 58 30 71 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 30 acgtacgtac gtcataggcg cgccaattaa tacgactcactatagggaga tttttttttt 60 tttttttttt v 71 31 96 DNA Artificial SequenceSynthetically generated oligonucleotide 31 acgtacgtac gtacgtacgtacgtcacgta cgtacgtcat aggcgcgcca attaatacga 60 ctcactatag ggagattttttttttttttt tttttv 96 32 33 DNA Artificial Sequence Syntheticallygenerated oligonucleotide 32 gcgccaatta tcgaaaaaaa aaaaaaaaaa aaa 33 3346 DNA Artificial Sequence Synthetically generated oligonucleotide 33attaatacga ctcactatag ggagattttt tttttttttt tttttv 46 34 52 DNAArtificial Sequence Synthetically generated oligonucleotide 34gcgccaatta atacgactca ctatagggag attttttttt tttttttttt tv 52 35 58 DNAArtificial Sequence Synthetically generated oligonucleotide 35ataggcgcgc caattaatac gactcactat agggagattt tttttttttt tttttttv 58

What is claimed is:
 1. A method of producing RNA replicates of sample nucleic acids, the method comprising: providing an insoluble support comprising attached oligonucleotides, wherein (1) the attached oligonucleotides comprise a promoter sequence and a target annealing sequence, wherein the target annealing sequence comprises a poly-thymidine tract and a 3′ terminal A, G, or C, and (2) the proximal end of the promoter sequence is spaced from the insoluble support by a distance greater than 10 nm, annealing sample nucleic acids to the attached oligonucleotides; constructing template nucleic acids by extending the attached oligonucleotides using a polymerase; and transcribing the template nucleic acids to produce RNA replicates of the sample nucleic acids.
 2. The method of claim 1 wherein the distance is between 10 to 150 nm.
 3. The method of claim 2 wherein the distance is between 10 and 50 nm.
 4. The method of claim 1 wherein the attached oligonucleotides each comprise a biotin moiety at the 5′ terminus, are attached to the insoluble support by a biotin/biotin-binding protein interaction, and the proximal end of the promoter is between 5 and 30 nucleotides from the 5′ terminus.
 5. The method of claim 4 wherein the proximal end of the promoter is between 6 and 18 nucleotides from the 5′ terminus.
 6. The method of claim 1 wherein the attached oligonucleotides are attached to the insoluble support by a polyethylene glycol linker which has between 8 and 16 units.
 7. The method of claim 1 wherein the attached oligonucleotides are covalently attached at their 5′ terminus, and the proximal end of the promoter is between 12 and 50 nucleotides from the 5′ terminus of each of the attached oligonucleotides.
 8. The method of claim 1 wherein the sample nucleic acids comprise RNA molecules.
 9. The method of claim 1 wherein the sample nucleic acids comprise DNA molecules.
 10. The method of claim 2 wherein the constructing comprises extending the attached oligonucleotide using an RNA-directed DNA polymerase to produce an extended stranded and synthesizing a DNA strand complementary to the extended strand to produce complementary strands, wherein the attached and complementary strands anneal, thereby providing the template nucleic acids.
 11. The method of claim 1 further comprising joining an adaptor that comprises a tag sequence to the double-stranded template.
 12. The method of claim 11 wherein the adaptor comprises double-stranded DNA.
 13. The method of claim 12 wherein the adaptor comprises a promoter sequence.
 14. The method of claim 1 wherein at least some of the attached oligonucleotides comprise a spacer region of low complexity or medium complexity.
 15. The method of claim 14 wherein the attached oligonucleotides further comprise a spacer region having a sequence absent from a sample of relevance.
 16. The method of claim 1 wherein the distance is sufficient to enable at least twice the yield of replicate RNAs as obtained using a distance of less than 2 nm between the proximal end of the promoter sequence and the insoluble support.
 17. A method of producing RNA replicates of sample nucleic acids, the method comprising: providing an insoluble support comprising attached template nucleic acids, wherein (i) each attached template nucleic acids comprise a promoter sequence and a target sequence, and (ii) the proximal end of the promoter sequence is spaced from the insoluble support by a predetermined distance wherein (a) the predetermined distance is between 10 to 150 nm; (b) the attached template nucleic acids comprise a biotin at its 5′ terminus, are attached to the insoluble support by a biotin/biotin-binding protein interaction, and the proximal end of the promoter is between 5 and 30 nucleotides from the 5′ terminus, (c) the attached template nucleic acids are attached to the insoluble support by a polyethylene glycol linker which has between 8 and 16 units, or (d) the attached template nucleic acids are covalently attached, and the proximal end of the promoter is between 12 and 50 nucleotides from the 5′ terminus of each of the oligonucleotides; and transcribing the template nucleic acids to produce RNA replicates of the sample nucleic acids.
 18. The method of claim 17 wherein the template nucleic acids further comprise a second promoter positioned to transcribe a nucleic acid segment located between the first and second promoters, each configured to transcribe a strand of the nucleic acid segment such that both strands of the nucleic acid segment are transcribed, and the method comprises transcribing the template nucleic acid using the first and second promoters to produce RNA complementary to each strand, and recovering double-stranded RNA for the nucleic acid segment.
 19. The method of claim 1 wherein the attached oligonucleotides are covalently attached.
 20. The method of claim 1 wherein the attached oligonucleotides are non-covalently attached.
 21. A method of archiving a sample of complex nucleic acids, the method comprising: providing a first insoluble support having 5′ attached oligonucleotide, wherein the attached oligonucleotide comprises a promoter sequence that is at least 4 nm from the insoluble support and a target annealing sequence comprising a poly-thymidine tract and a 3′ terminal A, G, or C; annealing a complex sample that comprises sample nucleic acids to the insoluble support; and producing template nucleic acids immobilized on the insoluble support that each include at least a segment of the sample nucleic acids, the immobilized templates representing the composition of the sample nucleic acids; transcribing the template nucleic acids from the insoluble support; archiving the insoluble support; and transcribing the template nucleic acids from the insoluble support.
 22. An insoluble support comprising a plurality of attached oligonucleotides, wherein (a) the attached oligonucleotides comprise a prokaryotic promoter sequence and a target annealing sequence, wherein each target annealing sequence of the plurality comprises a poly-thymidine tract and a 3′ terminal A, Q or C, (b) the target annealing sequence is 3′ of the promoter, (c) the oligonucleotide has an extendable 3′ terminus; and (d) the proximal end of the promoter sequence is spaced from the insoluble support by a distance greater than 10 nm.
 23. The support of claim 22 wherein the oligonucleotides are less than 80 nucleotides in length.
 24. The support of claim 22 wherein the support comprises a planar glass slide.
 25. The support of claim 22 wherein the attached oligonucleotides each comprise a attachment ligand 5′ of the promoter, are attached to the insoluble support by a protein-ligand interaction that binds the attachment ligand of the oligonucleotide to a protein on the support, and the proximal end of the promoter is between 5 and 30 nucleotides from the 5′ terminus.
 26. The support of claim 25 wherein the attachment ligand is biotin.
 27. The support of claim 26 wherein biotin moiety is attached to the 5′ terminus of the attached oligonucleotide.
 28. The support of claim 22 wherein the proximal end of the promoter is between 6 and 18 nucleotides from the 5′ terminus.
 29. The support of claim 22 wherein the attached oligonucleotides are covalently attached at an attachment site, and the proximal end of the promoter is between 12 and 50 nucleotides from the attachment site of each of the attached oligonucleotides.
 30. The support of claim 22 wherein the attached oligonucleotides are covalently attached at their 5′ terminus, and the proximal end of the promoter is between 12 and 50 nucleotides from the 5′ terminus of each of the attached oligonucleotides.
 31. An insoluble support comprising attached template nucleic acids, wherein (a) each attached template nucleic acids comprise a prokaryotic promoter sequence, a target annealing sequence, and a ligand, wherein each target annealing sequence of the plurality comprises a poly-thymidine tract and has a 3′ A, G, or C, (b) for each template nucleic acid, the promoter is located between the target sequence and ligand, (c) the template nucleic acids can be transcribed to produce RNA copies of each respective target sequence, (d) the ligand is bound to a ligand-binding protein immobilized on the support, and (e) the proximal end of the promoter sequence is spaced from the ligand between 5 and 30 nucleotides.
 32. The support of claim 31 wherein the ligand is biotin. 